U.S. patent application number 11/047257 was filed with the patent office on 2005-08-25 for phosphatase -encoding nucleic acids and methods of making and using them.
Invention is credited to Bylina, Edward, Lee, Edd, Mathur, Eric J., Short, Jay M..
Application Number | 20050186605 11/047257 |
Document ID | / |
Family ID | 34865011 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186605 |
Kind Code |
A1 |
Short, Jay M. ; et
al. |
August 25, 2005 |
Phosphatase -encoding nucleic acids and methods of making and using
them
Abstract
The invention relates to thermostable phosphatases and to
polynucleotides encoding the thermostable phosphatases. In addition
methods of designing new phosphatases and methods of use thereof
are also provided. The thermostable phosphatases have increased
activity and stability at increased pH and temperature.
Inventors: |
Short, Jay M.; (Del Mar,
CA) ; Mathur, Eric J.; (Carlsbad, CA) ; Lee,
Edd; (Del Mar, CA) ; Bylina, Edward;
(Henderson, CA) |
Correspondence
Address: |
DIVERSA CORPORATION
4955 DIRECTORS PLACE
SAN DIEGO
CA
92121
US
|
Family ID: |
34865011 |
Appl. No.: |
11/047257 |
Filed: |
January 31, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11047257 |
Jan 31, 2005 |
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09902525 |
Jul 9, 2001 |
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09902525 |
Jul 9, 2001 |
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09202681 |
Dec 23, 1999 |
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09202681 |
Dec 23, 1999 |
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PCT/US97/10784 |
Jun 19, 1997 |
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60033752 |
Jun 19, 1996 |
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Current U.S.
Class: |
435/6.14 ;
435/196; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/16 20130101 |
Class at
Publication: |
435/006 ;
536/023.2; 435/069.1; 435/196; 435/320.1; 435/325 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/16 |
Claims
What is claimed is:
1: An isolated or recombinant nucleic acid comprising (a) a
sequence having at least 70% sequence identity to SEQ ID NO:21, and
encoding a polypeptide having a phosphatase activity, or (b)
sequences complementary to (a).
2: The isolated or recombinant nucleic acid of claim 1, comprising
a sequence as set forth in SEQ ID NO:21, and sequences
complementary thereto.
3: An isolated or recombinant nucleic acid at least 20 residues in
length that hybridizes to a nucleic acid encoding a polypeptide
having a phosphatase activity, or its complementary sequence, under
conditions comprising a wash under conditions comprising (a) wash
in a solution comprising 0.1.times.SSC, 0.5% SDS for 15 to 30
minutes or (b) wash in a solution comprising 150 mM NaCl, 20 mM
Tris hydrochloride, pH 7.8, 1 mM Na.sub.2EDTA, 0.5% SDS, followed
by a 30 minute wash in fresh solution, wherein the nucleic acid
encoding a polypeptide having phosphatase activity has at least 70%
sequence identity to SEQ ID NO:21.
4: The isolated or recombinant nucleic acid of claim 3, wherein the
conditions comprise a wash under conditions comprising
0.1.times.SSC, 0.5% SDS at room temperature for 30 minutes to 1
hour.
5: The isolated or recombinant nucleic acid of claim 3, wherein the
conditions comprise a wash under conditions comprising 2.times.SSC,
0.1% SDS at room temperature for 15 minutes.
6: An isolated or recombinant nucleic acid comprising (a) a
sequence having at least 70% sequence identity to a nucleic acid
encoding a polypeptide having phosphatase activity and a sequence
as set forth in SEQ ID NO:30, or (b) a sequence complementary to
(a).
7: The isolated or recombinant nucleic acid of claim 6, wherein the
nucleic acid sequence has (a) at least 75% sequence identity to a
nucleic acid encoding a polypeptide having phosphatase activity and
having a sequence as set forth in SEQ ID NO:30.
8: The isolated or recombinant nucleic acid of claim 7, wherein the
nucleic acid sequence has at least 80% sequence identity to a
nucleic acid encoding a polypeptide having phosphatase activity and
having a sequence as set forth in SEQ ID NO:30.
9: The isolated or recombinant nucleic acid of claim 8, wherein the
nucleic acid sequence has at least 85% sequence identity to a
nucleic acid encoding a polypeptide having phosphatase activity and
having a sequence as set forth in SEQ ID NO:30.
10: The isolated or recombinant nucleic acid of claim 9, wherein
the nucleic acid sequence has at least 90% sequence identity to a
nucleic acid a encoding a polypeptide having phosphatase activity
and having a sequence as set forth in SEQ ID NO:30.
11: The isolated or recombinant nucleic acid of claim 10, wherein
the nucleic acid sequence has at least 95% sequence identity to a
nucleic acid encoding a polypeptide having phosphatase activity and
having a sequence as set forth in SEQ ID NO:30.
12. The isolated or recombinant nucleic acid of claim 11, wherein
the nucleic acid sequence has at least 97% sequence identity to a
nucleic acid encoding a polypeptide having phosphatase activity and
having a sequence as set forth in SEQ ID NO:30.
13: The isolated or recombinant nucleic acid of claim 12, wherein
the nucleic acid sequence has at least 99% sequence identity to a
nucleic acid encoding a polypeptide having phosphatase activity and
having a sequence as set forth in SEQ ID NO:30.
14: An isolated or recombinant nucleic acid comprising (a) a
sequence as set forth in SEQ ID NO:30 encoding a polypeptide having
phosphatase activity and comprising at least one conservative amino
acid substitution, or (b) a sequence complementary to (a).
15: The isolated or recombinant nucleic acid of claim 1, wherein
the sequence identity is determined by analysis with FASTA version
3.0t78 with the default parameters.
16: An isolated or recombinant nucleic acid encoding a polypeptide
having phosphatase activity and comprising at least 20 consecutive
bases of a sequence as set forth in SEQ ID NO:21, and sequences
complementary thereto.
17: An isolated or recombinant nucleic acid comprising (a) a
sequence encoding a polypeptide having phosphatase activity and
having at least 70% sequence identity to at least 20 consecutive
bases of a sequence as set forth in SEQ ID NO:21 as determined by
analysis with FASTA version 3.0t78 with the default parameters, or
(b) a sequence complementary to (a).
18: The isolated or recombinant nucleic acid of claim 17, wherein
the nucleic acid encodes a polypeptide having phosphatase activity
and has at least 75% sequence identity to at least 20 consecutive
bases of a sequence as set forth in SEQ ID NO:21 as determined by
analysis with FASTA version 3.0t78 with the default parameters.
19: The isolated or recombinant nucleic acid of claim 18, wherein
the nucleic acid encodes a polypeptide having phosphatase activity
and has at least 80% sequence identity to at least 20 consecutive
bases of a sequence as set forth in SEQ ID NO:21 as determined by
analysis with FASTA version 3.0t78 with the default parameters.
20: The isolated or recombinant nucleic acid of claim 19, wherein
the nucleic acid encodes a polypeptide having phosphatase activity
and has at least 85% sequence identity to at least 20 consecutive
bases of a sequence as set forth in SEQ ID NO:21 as determined by
analysis with FASTA version 3.0t78 with the default parameters.
21: The isolated or recombinant nucleic acid of claim 20, wherein
the nucleic acid encodes a polypeptide having phosphatase activity
and has at least 90% sequence identity to at least 20 consecutive
bases of a sequence as set forth in SEQ ID NO:21 as determined by
analysis with FASTA version 3.0t78 with the default parameters.
22: An isolated or recombinant nucleic acid comprising (a) a
sequence encoding a polypeptide having phosphatase activity
comprising at least 20 consecutive amino acids of a polypeptide
having a sequence as set forth in SEQ ID NO:30, or (b) a sequence
complementary to (a).
23: The isolated or recombinant nucleic acid of claim 22, wherein
the polypeptide having phosphatase activity has a sequence as set
forth in SEQ ID NO:30.
24: A method of producing a polypeptide having phosphatase activity
comprising at least 10 amino acids of a sequence as set forth in
SEQ ID NO:30, or a sequence encoded by a nucleic acid as set forth
in claim 1, comprising introducing the nucleic acid into a host
cell under conditions that allow expression of the polypeptide.
25: The method of claim 24, wherein the polypeptide having
phosphatase activity has a sequence as set forth in SEQ ID NO:30,
or a polypeptide having phosphatase activity encoded by a nucleic
acid as set forth in claim 1.
26: A nucleic acid probe for identifying or isolating a nucleic
acid encoding a polypeptide having phosphatase activity comprising
an oligonucleotide from about 10 to 50 nucleotides in length and
having an area of at least 20 contiguous nucleotides that has at
least 70% sequence identity to a nucleic acid target region
comprising a nucleic acid sequence as set forth in SEQ ID NO:21, or
its complementary sequence, and which hybridizes to the nucleic
acid target region to form a detectable target:probe duplex under
hybridization conditions comprising a wash under conditions
comprising 0.1.times.SSC, 0.5% SDS at room temperature for 30
minutes to 1 hour, or (b) wash in a solution comprising 150 mM
NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na.sub.2EDTA, 0.5%
SDS, followed by a 30 minute wash in fresh solution.
27: The probe of claim 26, wherein the oligonucleotide comprises a
DNA or an RNA.
28: The probe of claim 26, wherein the area has at least 25
contiguous nucleotides.
29: The probe of claim 26, wherein the area has at least 30
contiguous nucleotides.
30: The probe of claim 26, wherein the area has at least 35
contiguous nucleotides.
31: The probe of claim 26, wherein the area has at least 50
contiguous nucleotides.
32: The probe of claim 26, wherein the area has at least 75%
sequence identity to the nucleic acid target region.
33: The probe of claim 32, wherein the area has at least 80%
sequence identity to the nucleic acid target region.
34: The probe of claim 33, wherein the area has at least 85%
sequence identity to the nucleic acid target region.
35: The probe of claim 34, wherein the area has at least 90%
sequence identity to the nucleic acid target region.
36: The probe of claim 35, wherein the area has at least 95%
sequence identity to the nucleic acid target region.
37: The probe of claim 26, wherein the area is fully complementary
to the nucleic acid target region.
38: The probe of claim 26, wherein the oligonucleotide is 15-50
bases in length.
39: The probe of claim 26, wherein the probe further comprises a
detectable isotopic label.
40: The probe of claim 26, wherein the probe further comprises a
detectable non-isotopic label selected from the group consisting of
a fluorescent molecule, a chemiluminescent molecule, an enzyme, a
cofactor, an enzyme substrate, and a hapten.
41: A nucleic acid probe for isolation or identification of
phosphatase genes having a sequence which is the same as or fully
complementary to a sequence as set forth in SEQ ID NO:21
42: The isolated or recombinant nucleic acid of claim 3, wherein
the nucleic acid is at least 25 residues in length.
43: The isolated or recombinant nucleic acid of claim 42, wherein
the nucleic acid is at least 30 residues in length.
44: The isolated or recombinant nucleic acid of claim 43, wherein
the nucleic acid is at least 35 residues in length.
45: The isolated or recombinant nucleic acid of claim 44, wherein
the nucleic acid is at least 40 residues in length.
46: The isolated or recombinant nucleic acid of claim 45, wherein
the nucleic acid is at least 50 residues in length.
47: The isolated or recombinant nucleic acid of claim 46, wherein
the nucleic acid is at least 75 residues in length.
48: The isolated or recombinant nucleic acid of claim 47, wherein
the nucleic acid is at least 100 residues in length.
49: The isolated or recombinant nucleic acid of claim 48, wherein
the nucleic acid is at least 150 residues in length.
50: The isolated or recombinant nucleic acid of claim 17, wherein
the nucleic acid is at least 25 residues in length.
51: The isolated or recombinant nucleic acid of claim 50, wherein
the nucleic acid is at least 30 residues in length.
52: The isolated or recombinant nucleic acid of claim 51, wherein
the nucleic acid is at least 35 residues in length.
53: The isolated or recombinant nucleic acid of claim 52, wherein
the nucleic acid is at least 40 residues in length.
54: The isolated or recombinant nucleic acid of claim 53, wherein
the nucleic acid is at least 50 residues in length.
55: The isolated or recombinant nucleic acid of claim 54, wherein
the nucleic acid is at least 75 residues in length.
56: The isolated or recombinant nucleic acid of claim 55, wherein
the nucleic acid is at least 100 residues in length.
57: The isolated or recombinant nucleic acid of claim 56, wherein
the nucleic acid is at least 150 residues in length.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/202,681, filed Dec. 23, 1999, now pending;
which is the National Stage Application of US97/10784, filed Jun.
19, 1997; which claims priority to U.S. Provisional Application
Ser. No. 60/033,752, the contents of which are hereby incorporated
by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to enzymes, polynucleotides
encoding the enzymes, the use of such polynucleotides and
polypeptides, and more specifically to enzymes having thermostable
phosphatase activity.
BACKGROUND
[0003] Phosphatases are a group of enzymes that remove phosphate
groups from organophosphate ester compounds. There are numerous
phosphatases, including alkaline phosphatases, phosphodiesterases
and phytases.
[0004] Alkaline phosphatases are widely distributed enzymes and are
composed of a group of enzymes which hydrolyze organic phosphate
ester bonds at alkaline pH.
[0005] Phosphodiesterases are capable of hydrolyzing nucleic acids
by hydrolyzing the phosphodiester bridges of DNA and RNA. The
classification of phosphodiesterases depends upon which side of the
phosphodiester bridge is attacked. The 3' enzymes specifically
hydrolyze the ester linkage between the 3' carbon and the
phosphoric group whereas the 5' enzymes hydrolyze the ester linkage
between the phosphoric group and the 5' carbon of the
phosphodiester bridge. The best known of the class 3' enzymes is a
phosphodiesterase from the venom of the rattlesnake or from a
rustle's viper, which hydrolyses all the 3' bonds in either RNA or
DNA liberating nearly all the nucleotide units as nucleotide 5'
phosphates. This enzyme requires a free 3' hydroxyl group on the
terminal nucleotide residue and proceeds stepwise from that end of
the polynucleotide chain. This enzyme and all other nucleases which
attack only at the ends of the polynucleotide chains are called
exonucleases. The 5' enzymes are represented by a phosphodiesterase
from bovine spleen, also an exonuclease, which hydrolyses all the
5' linkages of both DNA and RNA and thus liberates only nucleoside
3' phosphates. It begins its attack at the end of the chain having
a free 3' hydroxyl group.
[0006] Phytases are enzymes which recently have been introduced to
commerce. The phytase enzyme removes phosphate from phytic acid
(inositol hexaphosphoric acid), a compound found in plants such as
corn, wheat and rice. The enzyme has commercial use for the
treatment of animal feed, making the inositol of the phytic acid
available for animal nutrition. Aspergillus ficuum and wheat are
sources of phytase. (Business Communications Co., Inc., 25 Van Zant
Street, Norwalk, Conn. 06855).
[0007] Phytase is used to improve the utilization of natural
phosphorus in animal feed. Use of phytase as a feed additive
enables the animal to metabolize a larger degree of its cereal
feed's natural mineral content thereby reducing or altogether
eliminating the need for synthetic phosphorus additives. More
important than the reduced need for phosphorus additives is the
corresponding reduction of phosphorus in pig and chicken waste.
Many European countries severely limit the amount of manure that
can be spread per acre due to concerns regarding phosphorus
contamination of ground water. This is highly important in northern
Europe, and will eventually be regulated throughout the remainder
of the European Continent and the United States as well. (Excerpts
from Business Trend Analysts, Inc., January 1994, Frost and
Sullivan Report 1995 and USDA on-line information.)
[0008] Alkaline phosphatase hydrolyzes monophosphate esters,
releasing an organic phosphate and the cognate alcohol compound. It
is non-specific with respect to the alcohol moiety and it is this
feature which accounts for the many uses of this enzyme. The enzyme
has a pH optimum between 9 and 10, however, it can also function at
neutral pH, (study of the enzyme industry conducted by Business
Communications Company, Inc., 25 Van Zant Street, Norwalk, Conn.
06855, 1995.).
[0009] Thermostable alkaline phosphatases are not irreversibly
inactivated even when heated to 60.degree. C. or more for brief
periods of time, as, for example, in the practice of hydrolyzing
monophosphate esters.
[0010] Alkaline phosphatases may be obtained from numerous
thermophilic organisms, such as Ammonifex degensii, Aquifex
pyrophilus, Archaeoglobus lithotrophicus, Methanococcus igneus,
Pyrolobus (a Crenarchaeota), Pyrococcus and Thermococcus, which are
mostly Eubacteria and Euryarchaeota. Many of these organisms grow
at temperatures up to about 103.degree. C. and are unable to grow
below 70.degree. C. These anaerobes are isolated from extreme
environments. For example, The Thermococcus CL-2 was isolated from
a worm residing on a "black smoker" sulfite structure.
[0011] Interest in alkaline phosphatases from thermophilic microbes
has increased recently due to their value for commercial
applications. Two sources of alkaline phosphatases dominate and
compete commercially: (i) animal, from bovine and calf intestinal
mucosa, and (ii) bacterial, from E. coli. Due to the high turnover
number of calf intestinal phosphatase, it is often selected as the
label in many enzyme immunoassays. The usefulness of calf alkaline
phosphatase, however, is limited by its inherently low
thermostability, which is even further compromised during the
chemical preparation of the enzyme: antibody conjugates. Bacterial
alkaline phosphatase is an alternative to calf alkaline phosphatase
due to bacterial alkaline phosphatase's extreme thermotolerance at
temperatures as high as 95.degree. C. (Tomazic-Allen, S. J.;
Recombinant Bacterial Phosphatase as an Immunodiagnostic Enzyme,
Annals D Biology Clinique, 49(5):287-90 (1991)), however, the
enzyme has a very low turnover number.
[0012] There is a need for novel phosphatase enzymes having
enhanced thermostability. This includes a need for thermostable
alkaline phosphatases whose enhanced thermostability is beneficial
in enzyme labeling processes and certain recombinant DNA
techniques, such as in the dephosphorylation of vector DNA prior to
insert DNA ligation. Recombinant phosphatase enzymes provide the
proteins in a format amenable to efficient production of pure
enzyme, which can be utilized in a variety of applications as
described herein. Accordingly, there is a need for the
characterization, amino acid sequencing, DNA sequencing, and
heterologous expression of thermostable phosphatase enzymes. The
present invention meets these need by providing DNA and amino acid
sequence information and expression and purification protocol for
thermostable phosphatase derived from several organisms.
[0013] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the invention is not entitled to antedate such disclosure by virtue
of prior invention.
SUMMARY OF THE INVENTION
[0014] The invention provides an isolated nucleic acid having a
sequence as set forth in SEQ ID Nos.: 19, 20, 21, 22, 23, 24, 25,
26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53 and variants
thereof having at least 50% sequence identity to SEQ ID Nos.: 19,
20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49,
51, 53 and encoding polypeptides having phosphatase activity.
[0015] One aspect of the invention is an isolated nucleic acid
having a sequence as set forth in SEQ ID Nos.: 19, 20, 21, 22, 23,
24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53
(hereinafter referred to as "Group A nucleic acid sequences"),
sequences substantially identical thereto, and sequences
complementary thereto.
[0016] Another aspect of the invention is an isolated nucleic acid
including at least 10 consecutive bases of a sequence as set forth
in Group A nucleic acid sequences, sequences substantially
identical thereto, and the sequences complementary thereto.
[0017] In yet another aspect, the invention provides an isolated
nucleic acid encoding a polypeptide having a sequence as set forth
in SEQ ID Nos.:28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46,
47, 48, 50, 52, 54, and variants thereof encoding a polypeptide
having phosphatase activity and having at least 50% sequence
identity to such sequences.
[0018] Another aspect of the invention is an isolated nucleic acid
encoding a polypeptide or a functional fragment thereof having a
sequence as set forth in SEQ ID Nos.:28, 29, 30, 31, 32, 33, 34,
35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, (hereinafter referred
to as "Group B amino acid sequences"), and sequences substantially
identical thereto.
[0019] Another aspect of the invention is an isolated nucleic acid
encoding a polypeptide having at least 10 consecutive amino acids
of a sequence as set forth in Group B amino acid sequences, and
sequences substantially identical thereto.
[0020] In yet another aspect, the invention provides a purified
polypeptide having a sequence as set forth in Group B amino acid
sequences, and sequences substantially identical thereto.
[0021] Another aspect of the invention is an isolated or purified
antibody that specifically binds to a polypeptide having a sequence
as set forth in Group B amino acid sequences, and sequences
substantially identical thereto.
[0022] Another aspect of the invention is an isolated or purified
antibody or binding fragment thereof, which specifically binds to a
polypeptide having at least 10 consecutive amino acids of one of
the polypeptides of Group B amino acid sequences, and sequences
substantially identical thereto.
[0023] Another aspect of the invention is a method of making a
polypeptide having a sequence as set forth in Group B amino acid
sequences, and sequences substantially identical thereto. The
method includes introducing a nucleic acid encoding the polypeptide
into a host cell, wherein the nucleic acid is operably linked to a
promoter, and culturing the host cell under conditions that allow
expression of the nucleic acid.
[0024] Another aspect of the invention is a method of making a
polypeptide having at least 10 amino acids of a sequence as set
forth in Group B amino acid sequences, and sequences substantially
identical thereto. The method includes introducing a nucleic acid
encoding the polypeptide into a host cell, wherein the nucleic acid
is operably linked to a promoter, and culturing the host cell under
conditions that allow expression of the nucleic acid, thereby
producing the polypeptide.
[0025] Another aspect of the invention is a method of generating a
variant including obtaining a nucleic acid having a sequence as set
forth in Group A nucleic acid sequences, sequences substantially
identical thereto, sequences complementary to the sequences of
Group A nucleic acid sequences, fragments comprising at least 30
consecutive nucleotides of the foregoing sequences, and changing
one or more nucleotides in the sequence to another nucleotide,
deleting one or more nucleotides in the sequence, or adding one or
more nucleotides to the sequence.
[0026] Another aspect of the invention is a computer readable
medium having stored thereon a sequence as set forth in Group A
nucleic acid sequences, and sequences substantially identical
thereto, or a polypeptide sequence as set forth in Group B amino
acid sequences, and sequences substantially identical thereto.
[0027] Another aspect of the invention is a computer system
including a processor and a data storage device wherein the data
storage device has stored thereon a sequence as set forth in Group
A nucleic acid sequences, and sequences substantially identical
thereto, or a polypeptide having a sequence as set forth in Group B
amino acid sequences, and sequences substantially identical
thereto.
[0028] Another aspect of the invention is a method for comparing a
first sequence to a reference sequence wherein the first sequence
is a nucleic acid having a sequence as set forth in Group A nucleic
acid sequences, and sequences substantially identical thereto, or a
polypeptide code of Group B amino acid sequences, and sequences
substantially identical thereto. The method includes reading the
first sequence and the reference sequence through use of a computer
program which compares sequences; and determining differences
between the first sequence and the reference sequence with the
computer program.
[0029] Another aspect of the invention is a method for identifying
a feature in a sequence as set forth in Group A nucleic acid
sequences, and sequences substantially identical thereto, or a
polypeptide having a sequence as set forth in Group B amino acid
sequences, and sequences substantially identical thereto, including
reading the sequence through the use of a computer program which
identifies features in sequences; and identifying features in the
sequence with the computer program.
[0030] Another aspect of the invention is an assay for identifying
fragments or variants of Group B amino acid sequences, and
sequences substantially identical thereto, which retain the
enzymatic function of the polypeptides of Group B amino acid
sequences, and sequences substantially identical thereto. The assay
includes contacting the polypeptide of Group B amino acid
sequences, sequences substantially identical thereto, or
polypeptide fragment or variant with a substrate molecule under
conditions which allow the polypeptide fragment or variant to
function, and detecting either a decrease in the level of substrate
or an increase in the level of the specific reaction product of the
reaction between the polypeptide and substrate thereby identifying
a fragment or variant of such sequences.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The following drawings are illustrative of embodiments of
the invention and are not meant to limit the scope of the invention
as encompassed by the claims.
[0032] FIG. 1 is a block diagram of a computer system.
[0033] FIG. 2 is a flow diagram illustrating one embodiment of a
process for comparing a new nucleotide or protein sequence with a
database of sequences in order to determine the homology levels
between the new sequence and the sequences in the database.
[0034] FIG. 3 is a flow diagram illustrating one embodiment of a
process in a computer for determining whether two sequences are
homologous.
[0035] FIG. 4 is a flow diagram illustrating one embodiment of an
identifier process 300 for detecting the presence of a feature in a
sequence.
[0036] FIG. 5 is an illustration of the full-length DNA and
corresponding deduded amino acid sequence of Ammonifex degensii KC4
of the present invention. Sequencing was performed using a 378
automated DNA sequence for all sequences of the present invention
(Applied Biosystems, Inc. Foster City, Calif.).
[0037] FIG. 6 is an illustration of the full-length DNA and
corresponding deduded amino acid sequence of Methanococcus igneus
Ko 15.
[0038] FIG. 7 is an illustration of the full-length DNA and
corresponding deduded amino acid sequence of Thermococcus
alcaliphilus AEDII12RA.
[0039] FIG. 8 is an illustration of the full-length DNA and
corresponding deduded amino acid sequence of Thermococcus
celer.
[0040] FIG. 9 is an illustration of the full-length DNA and
corresponding deduded amino acid sequence of Thermococcus
GU5L5.
[0041] FIG. 10 is an illustration of the full-length DNA and
corresponding deduded amino acid sequence of OC9a.
[0042] FIG. 11 is an illustration of the full-length DNA and
corresponding deduded amino acid sequence of M11TL.
[0043] FIG. 12 is an illustration of the full-length DNA and
corresponding deduded amino acid sequence of Thermococcus CL-2.
[0044] FIG. 13 is an illustration of the full-length DNA and
corresponding deduded amino acid sequence of Aquifex VF-5.
DETAILED DESCRIPTION OF THE INVENTION
[0045] The present invention relates to phosphatases and
polynucleotides encoding them. As used herein, the term
"phosphatase" encompasses enzymes having hydrolase activity, for
example, enzymes capable of hydrolyzing glycosidic linkages present
in starch.
[0046] The polynucleotides of the invention have been identified as
encoding polypeptides having phosphatase activity.
DEFINITIONS
[0047] The phrases "nucleic acid" or "nucleic acid sequence" as
used herein refer to an oligonucleotide, nucleotide,
polynucleotide, or to a fragment of any of these, to DNA or RNA of
genomic or synthetic origin which may be single-stranded or
double-stranded and may represent a sense or antisense strand, to
peptide nucleic acid (PNA), or to any DNA-like or RNA-like
material, natural or synthetic in origin.
[0048] A "coding sequence of" or a "nucleotide sequence encoding" a
particular polypeptide or protein, is a nucleic acid sequence which
is transcribed and translated into a polypeptide or protein when
placed under the control of appropriate regulatory sequences.
[0049] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as, where
applicable, intervening sequences (introns) between individual
coding segments (exons).
[0050] "Amino acid" or "amino acid sequence" as used herein refer
to an oligopeptide, peptide, polypeptide, or protein sequence, or
to a fragment, portion, or subunit of any of these, and to
naturally occurring or synthetic molecules.
[0051] The term "polypeptide" as used herein, refers to amino acids
joined to each other by peptide bonds or modified peptide bonds,
i.e., peptide isosteres, and may contain modified amino acids other
than the 20 gene-encoded amino acids. The polypeptides may be
modified by either natural processes, such as post-translational
processing, or by chemical modification techniques which are well
known in the art. Modifications can occur anywhere in the
polypeptide, including the peptide backbone, the amino acid
side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in
the same or varying degrees at several sites in a given
polypeptide. Also a given polypeptide may have many types of
modifications. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of a phosphytidylinositol,
cross-linking cyclization, disulfide bond formation, demethylation,
formation of covalent cross-links, formation of cysteine, formation
of pyroglutamate, formylation, gamma-carboxylation, glycosylation,
GPI anchor formation, hydroxylation, iodination, methylation,
myristolyation, oxidation, pergylation, proteolytic processing,
phosphorylation, prenylation, racemization, selenoylation,
sulfation, and transfer-RNA mediated addition of amino acids to
protein such as arginylation. (See Creighton, T. E.,
Proteins--Structure and Molecular Properties 2nd Ed., W. H. Freeman
and Company, New York (1993); Posttranslational Covalent
Modification of Proteins, B. C. Johnson, Ed., Academic Press, New
York, pp. 1-12 (1983)).
[0052] As used herein, the term "isolated" means that the material
is removed from its original environment (e.g., the natural
environment if it is naturally occurring). For example, a
naturally-occurring polynucleotide or polypeptide present in a
living animal is not isolated, but the same polynucleotide or
polypeptide, separated from some or all of the coexisting materials
in the natural system, is isolated. Such polynucleotides could be
part of a vector and/or such polynucleotides or polypeptides could
be part of a composition, and still be isolated in that such vector
or composition is not part of its natural environment.
[0053] As used herein, the term "purified" does not require
absolute purity; rather, it is intended as a relative definition.
Individual nucleic acids obtained from a library have been
conventionally purified to electrophoretic homogeneity. The
sequences obtained from these clones could not be obtained directly
either from the library or from total human DNA. The purified
nucleic acids of the invention have been purified from the
remainder of the genomic DNA in the organism by at least
10.sup.4-10.sup.6 fold. However, the term "purified" also includes
nucleic acids which have been purified from the remainder of the
genomic DNA or from other sequences in a library or other
environment by at least one order of magnitude, typically two or
three orders, and more typically four or five orders of
magnitude.
[0054] As used herein, the term "recombinant" means that the
nucleic acid is adjacent to a "backbone" nucleic acid to which it
is not adjacent in its natural environment. Additionally, to be
"enriched" the nucleic acids will represent 5% or more of the
number of nucleic acid inserts in a population of nucleic acid
backbone molecules. Backbone molecules according to the invention
include nucleic acids such as expression vectors, self-replicating
nucleic acids, viruses, integrating nucleic acids, and other
vectors or nucleic acids used to maintain or manipulate a nucleic
acid insert of interest. Typically, the enriched nucleic acids
represent 15% or more of the number of nucleic acid inserts in the
population of recombinant backbone molecules. More typically, the
enriched nucleic acids represent 50% or more of the number of
nucleic acid inserts in the population of recombinant backbone
molecules. In a one embodiment, the enriched nucleic acids
represent 90% or more of the number of nucleic acid inserts in the
population of recombinant backbone molecules.
[0055] "Recombinant" polypeptides or proteins refer to polypeptides
or proteins produced by recombinant DNA techniques; i.e., produced
from cells transformed by an exogenous DNA construct encoding the
desired polypeptide or protein. "Synthetic" polypeptides or protein
are those prepared by chemical synthesis. Solid-phase chemical
peptide synthesis methods can also be used to synthesize the
polypeptide or fragments of the invention. Such method have been
known in the art since the early 1960's (Merrifield, R. B., J. Am.
Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,
J. D., Solid Phase Peptide Synthesis 2nd Ed., Pierce Chemical Co.,
Rockford, Ill., pp. 11-12)) and have recently been employed in
commercially available laboratory peptide design and synthesis kits
(Cambridge Research Biochemicals). Such commercially available
laboratory kits have generally utilized the teachings of H. M.
Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and
provide for synthesizing peptides upon the tips of a multitude of
"rods" or "pins" all of which are connected to a single plate. When
such a system is utilized, a plate of rods or pins is inverted and
inserted into a second plate of corresponding wells or reservoirs,
which contain solutions for attaching or anchoring an appropriate
amino acid to the pin's or rod's tips. By repeating such a process
step, i.e., inverting and inserting the rod's and pin's tips into
appropriate solutions, amino acids are built into desired peptides.
In addition, a number of available FMOC peptide synthesis systems
are available. For example, assembly of a polypeptide or fragment
can be carried out on a solid support using an Applied Biosystems,
Inc. Model 431A automated peptide synthesizer. Such equipment
provides ready access to the peptides of the invention, either by
direct synthesis or by synthesis of a series of fragments that can
be coupled using other known techniques.
[0056] A promoter sequence is "operably linked to" a coding
sequence when RNA polymerase which initiates transcription at the
promoter will transcribe the coding sequence into mRNA.
[0057] "Plasmids" are designated by a lower case "p" preceded
and/or followed by capital letters and/or numbers. The starting
plasmids herein are either commercially available, publicly
available on an unrestricted basis, or can be constructed from
available plasmids in accord with published procedures. In
addition, equivalent plasmids to those described herein are known
in the art and will be apparent to the ordinarily skilled
artisan.
[0058] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 .mu.g of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 .mu.l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 .mu.g of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation
times of about 1 hour at 37.degree. C. are ordinarily used, but may
vary in accordance with the supplier's instructions. After
digestion, gel electrophoresis may be performed to isolate the
desired fragment.
[0059] "Oligonucleotide" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide without adding a phosphate with an ATP in
the presence of a kinase. A synthetic oligonucleotide will ligate
to a fragment that has not been dephosphorylated.
[0060] The phrase "substantially identical" in the context of two
nucleic acids or polypeptides, refers to two or more sequences that
have at least 50%, 60%, 70%, 80%, and in some aspects 90-95%
nucleotide or amino acid residue identity, when compared and
aligned for maximum correspondence, as measured using one of the
known sequence comparison algorithms or by visual inspection.
Typically, the substantial identity exists over a region of at
least about 100 residues, and most commonly the sequences are
substantially identical over at least about 150-200 residues. In
some embodiments, the sequences are substantially identical over
the entire length of the coding regions.
[0061] Additionally a "substantially identical" amino acid sequence
is a sequence that differs from a reference sequence by one or more
conservative or non-conservative amino acid substitutions,
deletions, or insertions, particularly when such a substitution
occurs at a site that is not the active site of the molecule, and
provided that the polypeptide essentially retains its functional
properties. A conservative amino acid substitution, for example,
substitutes one amino acid for another of the same class (e.g.,
substitution of one hydrophobic amino acid, such as isoleucin,
valine, leucine, or methionine, for another, or substitution of one
polar amino acid for another, such as substitution of arginine for
lysine, glutamic acid for aspartic acid or glutamine for
asparagine). One or more amino acids can be deleted, for example,
from a phosphatase polypeptide, resulting in modification of the
structure of the polypeptide, without significantly altering its
biological activity. For example, amino- or carboxyl-terminal amino
acids that are not required for phosphatase biological activity can
be removed. Modified polypeptide sequences of the invention can be
assayed for phosphatase biological activity by any number of
methods, including contacting the modified polypeptide sequence
with a phosphatase substrate and determining whether the modified
polypeptide decreases the amount of specific substrate in the assay
or increases the bioproducts of the enzymatic reaction of a
functional phosphatase polypeptide with the substrate.
[0062] "Fragments" as used herein are a portion of a naturally
occurring protein which can exist in at least two different
conformations. Fragments can have the same or substantially the
same amino acid sequence as the naturally occurring protein.
"Substantially the same" means that an amino acid sequence is
largely, but not entirely, the same, but retains at least one
functional activity of the sequence to which it is related. In
general two amino acid sequences are "substantially the same" or
"substantially homologous" if they are at least about 85%
identical. Fragments which have different three dimensional
structures as the naturally occurring protein are also included. An
example of this, is a "pro-form" molecule, such as a low activity
proprotein that can be modified by cleavage to produce a mature
enzyme with significantly higher activity.
[0063] "Hybridization" refers to the process by which a nucleic
acid strand joins with a complementary strand through base pairing.
Hybridization reactions can be sensitive and selective so that a
particular sequence of interest can be identified even in samples
in which it is present at low concentrations. Suitably stringent
conditions can be defined by, for example, the concentrations of
salt or formamide in the prehybridization and hybridization
solutions, or by the hybridization temperature, and are well known
in the art. In particular, stringency can be increased by reducing
the concentration of salt, increasing the concentration of
formamide, or raising the hybridization temperature.
[0064] For example, hybridization under high stringency conditions
could occur in about 50% formamide at about 37.degree. C. to
42.degree. C. Hybridization could occur under reduced stringency
conditions in about 35% to 25% formamide at about 30.degree. C. to
35.degree. C. In particular, hybridization could occur under high
stringency conditions at 42.degree. C. in 50% formamide,
5.times.SSPE, 0.3% SDS, and 200 n/ml sheared and denatured salmon
sperm DNA. Hybridization could occur under reduced stringency
conditions as described above, but in 35% formamide at a reduced
temperature of 35.degree. C. The temperature range corresponding to
a particular level of stringency can be further narrowed by
calculating the purine to pyrimidine ratio of the nucleic acid of
interest and adjusting the temperature accordingly. Variations on
the above ranges and conditions are well known in the art.
[0065] The term "variant" refers to polynucleotides or polypeptides
of the invention modified at one or more base pairs, codons,
introns, exons, or amino acid residues (respectively) yet still
retain the biological activity of a phosphatase of the invention.
Variants can be produced by any number of means included methods
such as, for example, error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, GSSM and any
combination thereof.
[0066] Enzymes are highly selective catalysts. Their hallmark is
the ability to catalyze reactions with exquisite stereo-, regio-,
and chemo- selectivities that are unparalleled in conventional
synthetic chemistry. Moreover, enzymes are remarkably versatile.
They can be tailored to function in organic solvents, operate at
extreme pHs (for example, high pHs and low pHs) extreme
temperatures (for example, high temperatures and low temperatures),
extreme salinity levels (for example, high salinity and low
salinity), and catalyze reactions with compounds that are
structurally unrelated to their natural, physiological
substrates.
[0067] Enzymes are reactive toward a wide range of natural and
unnatural substrates, thus enabling the modification of virtually
any organic lead compound. Moreover, unlike traditional chemical
catalysts, enzymes are highly enantio- and regio-selective. The
high degree of functional group specificity exhibited by enzymes
enables one to keep track of each reaction in a synthetic sequence
leading to a new active compound. Enzymes are also capable of
catalyzing many diverse reactions unrelated to their physiological
function in nature. For example, peroxidases catalyze the oxidation
of phenols by hydrogen peroxide. Peroxidases can also catalyze
hydroxylation reactions that are not related to the native function
of the enzyme. Other examples are proteases which catalyze the
breakdown of polypeptides. In organic solution some proteases can
also acylate sugars, a function unrelated to the native function of
these enzymes.
[0068] The present invention exploits the unique catalytic
properties of enzymes. Whereas the use of biocatalysts (i.e.,
purified or crude enzymes, non-living or living cells) in chemical
transformations normally requires the identification of a
particular biocatalyst that reacts with a specific starting
compound, the present invention uses selected biocatalysts and
reaction conditions that are specific for functional groups that
are present in many starting compounds.
[0069] Each biocatalyst is specific for one functional group, or
several related functional groups, and can react with many starting
compounds containing this functional group.
[0070] The biocatalytic reactions produce a population of
derivatives from a single starting compound. These derivatives can
be subjected to another round of biocatalytic reactions to produce
a second population of derivative compounds. Thousands of
variations of the original compound can be produced with each
iteration of biocatalytic derivatization.
[0071] Enzymes react at specific sites of a starting compound
without affecting the rest of the molecule, a process which is very
difficult to achieve using traditional chemical methods. This high
degree of biocatalytic specificity provides the means to identify a
single active compound within the library. The library is
characterized by the series of biocatalytic reactions used to
produce it, a so-called "biosynthetic history". Screening the
library for biological activities and tracing the biosynthetic
history identifies the specific reaction sequence producing the
active compound. The reaction sequence is repeated and the
structure of the synthesized compound determined. This mode of
identification, unlike other synthesis and screening approaches,
does not require immobilization technologies, and compounds can be
synthesized and tested free in solution using virtually any type of
screening assay. It is important to note, that the high degree of
specificity of enzyme reactions on functional groups allows for the
"tracking" of specific enzymatic reactions that make up the
biocatalytically produced library.
[0072] Many of the procedural steps are performed using robotic
automation enabling the execution of many thousands of biocatalytic
reactions and screening assays per day as well as ensuring a high
level of accuracy and reproducibility. As a result, a library of
derivative compounds can be produced in a matter of weeks which
would take years to produce using current chemical methods. (For
further teachings on modification of molecules, including small
molecules, see PCT/US94/09174, herein incorporated by reference in
its entirety).
[0073] In one aspect, the present invention provides a
non-stochastic method termed synthetic gene reassembly, that is
somewhat related to stochastic shuffling, save that the nucleic
acid building blocks are not shuffled or concatenated or chimerized
randomly, but rather are assembled non-stochastically.
[0074] The synthetic gene reassembly method does not depend on the
presence of a high level of homology between polynucleotides to be
shuffled. The invention can be used to non-stochastically generate
libraries (or sets) of progeny molecules comprised of over
10.sup.100 different chimeras. Conceivably, synthetic gene
reassembly can even be used to generate libraries comprised of over
10.sup.1000 different progeny chimeras.
[0075] Thus, in one aspect, the invention provides a non-stochastic
method of producing a set of finalized chimeric nucleic acid
molecules having an overall assembly order that is chosen by
design, which method is comprised of the steps of generating by
design a plurality of specific nucleic acid building blocks having
serviceable mutually compatible ligatable ends, and assembling
these nucleic acid building blocks, such that a designed overall
assembly order is achieved.
[0076] The mutually compatible ligatable ends of the nucleic acid
building blocks to be assembled are considered to be "serviceable"
for this type of ordered assembly if they enable the building
blocks to be coupled in predetermined orders. Thus, in one aspect,
the overall assembly order in which the nucleic acid building
blocks can be coupled is specified by the design of the ligatable
ends and, if more than one assembly step is to be used, then the
overall assembly order in which the nucleic acid building blocks
can be coupled is also specified by the sequential order of the
assembly step(s). In a one embodiment of the invention, the
annealed building pieces are treated with an enzyme, such as a
ligase (e.g., T4 DNA ligase) to achieve covalent bonding of the
building pieces.
[0077] In a another embodiment, the design of nucleic acid building
blocks is obtained upon analysis of the sequences of a set of
progenitor nucleic acid templates that serve as a basis for
producing a progeny set of finalized chimeric nucleic acid
molecules. These progenitor nucleic acid templates thus serve as a
source of sequence information that aids in the design of the
nucleic acid building blocks that are to be mutagenized, i.e.
chimerized or shuffled.
[0078] In one exemplification, the invention provides for the
chimerization of a family of related genes and their encoded family
of related products. In a particular exemplification, the encoded
products are enzymes. The phosphatases of the present invention can
be mutagenized in accordance with the methods described herein.
[0079] Thus according to one aspect of the invention, the sequences
of a plurality of progenitor nucleic acid templates (e.g.,
polynucleotides of Group A nucleic acid sequences) are aligned in
order to select one or more demarcation points, which demarcation
points can be located at an area of homology. The demarcation
points can be used to delineate the boundaries of nucleic acid
building blocks to be generated. Thus, the demarcation points
identified and selected in the progenitor molecules serve as
potential chimerization points in the assembly of the progeny
molecules.
[0080] Typically a serviceable demarcation point is an area of
homology (comprised of at least one homologous nucleotide base)
shared by at least two progenitor templates, but the demarcation
point can be an area of homology that is shared by at least half of
the progenitor templates, at least two thirds of the progenitor
templates, at least three fourths of the progenitor templates, and
preferably at almost all of the progenitor templates. Even more
preferably still a serviceable demarcation point is an area of
homology that is shared by all of the progenitor templates.
[0081] In a one embodiment, the gene reassembly process is
performed exhaustively in order to generate an exhaustive library.
In other words, all possible ordered combinations of the nucleic
acid building blocks are represented in the set of finalized
chimeric nucleic acid molecules. At the same time, the assembly
order (i.e. the order of assembly of each building block in the 5'
to 3 sequence of each finalized chimeric nucleic acid) in each
combination is by design (or non-stochastic). Because of the
non-stochastic nature of the method, the possibility of unwanted
side products is greatly reduced.
[0082] In another embodiment, the method provides that the gene
reassembly process is performed systematically, for example to
generate a systematically compartmentalized library, with
compartments that can be screened systematically, e.g., one by one.
In other words the invention provides that, through the selective
and judicious use of specific nucleic acid building blocks, coupled
with the selective and judicious use of sequentially stepped
assembly reactions, an experimental design can be achieved where
specific sets of progeny products are made in each of several
reaction vessels. This allows a systematic examination and
screening procedure to be performed. Thus, it allows a potentially
very large number of progeny molecules to be examined
systematically in smaller groups.
[0083] Because of its ability to perform chimerizations in a manner
that is highly flexible yet exhaustive and systematic as well,
particularly when there is a low level of homology among the
progenitor molecules, the instant invention provides for the
generation of a library (or set) comprised of a large number of
progeny molecules. Because of the non-stochastic nature of the
instant gene reassembly invention, the progeny molecules generated
preferably comprise a library of finalized chimeric nucleic acid
molecules having an overall assembly order that is chosen by
design. In a particularly embodiment, such a generated library is
comprised of greater than 10.sup.3 to greater than 10.sup.1000
different progeny molecular species.
[0084] In one aspect, a set of finalized chimeric nucleic acid
molecules, produced as described is comprised of a polynucleotide
encoding a polypeptide. According to one embodiment, this
polynucleotide is a gene, which may be a man-made gene. According
to another embodiment, this polynucleotide is a gene pathway, which
may be a man-made gene pathway. The invention provides that one or
more man-made genes generated by the invention may be incorporated
into a man-made gene pathway, such as pathway operable in a
eukaryotic organism (including a plant).
[0085] In another exemplification, the synthetic nature of the step
in which the building blocks are generated allows the design and
introduction of nucleotides (e.g., one or more nucleotides, which
may be, for example, codons or introns or regulatory sequences)
that can later be optionally removed in an in vitro process (e.g.,
by mutagenesis) or in an in vivo process (e.g., by utilizing the
gene splicing ability of a host organism). It is appreciated that
in many instances the introduction of these nucleotides may also be
desirable for many other reasons in addition to the potential
benefit of creating a serviceable demarcation point.
[0086] Thus, according to another embodiment, the invention
provides that a nucleic acid building block can be used to
introduce an intron. Thus, the invention provides that functional
introns may be introduced into a man-made gene of the invention.
The invention also provides that functional introns may be
introduced into a man-made gene pathway of the invention.
Accordingly, the invention provides for the generation of a
chimeric polynucleotide that is a man-made gene containing one (or
more) artificially introduced intron(s).
[0087] Accordingly, the invention also provides for the generation
of a chimeric polynucleotide that is a man-made gene pathway
containing one (or more) artificially introduced intron(s).
Preferably, the artificially introduced intron(s) are functional in
one or more host cells for gene splicing much in the way that
naturally-occurring introns serve functionally in gene splicing.
The invention provides a process of producing man-made
intron-containing polynucleotides to be introduced into host
organisms for recombination and/or splicing.
[0088] A man-made gene produced using the invention can also serve
as a substrate for recombination with another nucleic acid.
Likewise, a man-made gene pathway produced using the invention can
also serve as a substrate for recombination with another nucleic
acid. In a preferred instance, the recombination is facilitated by,
or occurs at, areas of homology between the man-made,
intron-containing gene and a nucleic acid, which serves as a
recombination partner. In a particularly preferred instance, the
recombination partner may also be a nucleic acid generated by the
invention, including a man-made gene or a man-made gene pathway.
Recombination may be facilitated by or may occur at areas of
homology that exist at the one (or more) artificially introduced
intron(s) in the man-made gene.
[0089] The synthetic gene reassembly method of the invention
utilizes a plurality of nucleic acid building blocks, each of which
preferably has two ligatable ends. The two ligatable ends on each
nucleic acid building block may be two blunt ends (i.e. each having
an overhang of zero nucleotides), or preferably one blunt end and
one overhang, or more preferably still two overhangs.
[0090] A useful overhang for this purpose may be a 3' overhang or a
5' overhang. Thus, a nucleic acid building block may have a 3'
overhang or alternatively a 5' overhang or alternatively two 3'
overhangs or alternatively two 5' overhangs. The overall order in
which the nucleic acid building blocks are assembled to form a
finalized chimeric nucleic acid molecule is determined by
purposeful experimental design and is not random.
[0091] According to one preferred embodiment, a nucleic acid
building block is generated by chemical synthesis of two
single-stranded nucleic acids (also referred to as single-stranded
oligos) and contacting them so as to allow them to anneal to form
a-double-stranded nucleic acid building block.
[0092] A double-stranded nucleic acid building block can be of
variable size. The sizes of these building blocks can be small or
large. Preferred sizes for building block range from 1 base pair
(not including any overhangs) to 100,000 base pairs (not including
any overhangs). Other preferred size ranges are also provided,
which have lower limits of from 1 bp to 10,000 bp (including every
integer value in between), and upper limits of from 2 bp to 100,000
bp (including every integer value in between).
[0093] Many methods exist by which a double-stranded nucleic acid
building block can be generated that is serviceable for the
invention; and these are known in the art and can be readily
performed by the skilled artisan.
[0094] According to one embodiment, a double-stranded nucleic acid
building block is generated by first generating two single stranded
nucleic acids and allowing them to anneal to form a double-stranded
nucleic acid building block. The two strands of a double-stranded
nucleic acid building block may be complementary at every
nucleotide apart from any that form an overhang; thus containing no
mismatches, apart from any overhang(s). According to another
embodiment, the two strands of a double-stranded nucleic acid
building block are complementary at fewer than every nucleotide
apart from any that form an overhang. Thus, according to this
embodiment, a double-stranded nucleic acid building block can be
used to introduce codon degeneracy. Preferably the codon degeneracy
is introduced using the site-saturation mutagenesis described
herein, using one or more N,N,G/T cassettes or alternatively using
one or more N,N,N cassettes.
[0095] The in vivo recombination method of the invention can be
performed blindly on a pool of unknown hybrids or alleles of a
specific polynucleotide or sequence. However, it is not necessary
to know the actual DNA or RNA sequence of the specific
polynucleotide.
[0096] The approach of using recombination within a mixed
population of genes can be useful for the generation of any useful
proteins, for example, interleukin I, antibodies, tPA and growth
hormone. This approach may be used to generate proteins having
altered specificity or activity. The approach may also be useful
for the generation of hybrid nucleic acid sequences, for example,
promoter regions, introns, exons, enhancer sequences, 31
untranslated regions or 51 untranslated regions of genes. Thus this
approach may be used to generate genes having increased rates of
expression. This approach may also be useful in the study of
repetitive DNA sequences. Finally, this approach may be useful to
mutate ribozymes or aptamers.
[0097] In one aspect the invention described herein is directed to
the use of repeated cycles of reductive reassortinent,
recombination and selection which allow for the directed molecular
evolution of highly complex linear sequences, such as DNA, RNA or
proteins thorough recombination.
[0098] In vivo shuffling of molecules is useful in providing
variants and can be performed utilizing the natural property of
cells to recombine multimers. While recombination in vivo has
provided the major natural route to molecular diversity, genetic
recombination remains a relatively complex process that involves 1)
the recognition of homologies; 2) strand cleavage, strand invasion,
and metabolic steps leading to the production of recombinant
chiasma; and finally 3) the resolution of chiasma into discrete
recombined molecules. The formation of the chiasma requires the
recognition of homologous sequences.
[0099] In another embodiment, the invention includes a method for
producing a hybrid polynucleotide from at least a first
polynucleotide and a second polynucleotide. The invention can be
used to produce a hybrid polynucleotide by introducing at least a
first polynucleotide and a second polynucleotide which share at
least one region of partial sequence homology into a suitable host
cell. The regions of partial sequence homology promote processes
which result in sequence reorganization producing a hybrid
polynucleotide. The term "hybrid polynucleotide", as used herein,
is any nucleotide sequence which results from the method of the
present invention and contains sequence from at least two original
polynucleotide sequences. Such hybrid polynucleotides can result
from intermolecular recombination events which promote sequence
integration between DNA molecules. In addition, such hybrid
polynucleotides can result from intramolecular reductive
reassortment processes which utilize repeated sequences to alter a
nucleotide sequence within a DNA molecule.
[0100] The invention provides a means for generating hybrid
polynucleotides which may encode biologically active hybrid
polypeptides (e.g., hybrid phosphatases). In one aspect, the
original-polynucleotides encode biologically active polypeptides.
The method of the invention produces new hybrid polypeptides by
utilizing cellular processes which integrate the sequence of the
original polynucleotides such that the resulting hybrid
polynucleotide encodes a polypeptide demonstrating activities
derived from the original biologically active polypeptides. For
example, the original polynucleotides may encode a particular
enzyme from different microorganisms. An enzyme encoded by a first
polynucleotide from one organism or variant may, for example,
function effectively under a particular environmental condition,
e.g. high salinity. An enzyme encoded by a second polynucleotide
from a different organism or variant may function effectively under
a different environmental condition, such as extremely high
temperatures. A hybrid polynucleotide containing sequences from the
first and second original polynucleotides may encode an enzyme
which exhibits characteristics of both enzymes encoded by the
original polynucleotides. Thus, the enzyme encoded by the hybrid
polynucleotide may function effectively under environmental
conditions shared by each of the enzymes encoded by the first and
second polynucleotides, e.g., high salinity and extreme
temperatures.
[0101] Enzymes encoded by the polynucleotides of the invention
include, but are not limited to, hydrolases, such as phosphatases.
A hybrid polypeptide resulting from the method of the invention may
exhibit specialized enzyme activity not displayed in the original
enzymes. For example, following recombination and/or reductive
reassortment of polynucleotides encoding hydrolase activities, the
resulting hybrid polypeptide encoded by a hybrid polynucleotide can
be screened for specialized hydrolase activities obtained from each
of the original enzymes, i.e. the type of bond on which the
hydrolase acts and the temperature at which the hydrolase
functions. Thus, for example, the hydrolase may be screened to
ascertain those chemical functionalities which distinguish the
hybrid hydrolase from the original hydrolases, such as: (a) amide
(peptide bonds), i.e., proteases; (b) ester bonds, i.e., esterases
and lipases; (c) acetals, i.e., glycosidases and, for example, the
temperature, pH or salt concentration at which the hybrid
polypeptide functions.
[0102] Sources of the original polynucleotides may be isolated from
individual organisms ("isolates"), collections of organisms that
have been grown in defined media ("enrichment cultures"), or,
uncultivated organisms ("environmental samples"). The use of a
culture-independent approach to derive polynucleotides encoding
novel bioactivities from environmental samples is most preferable
since it allows one to access untapped resources of
biodiversity.
[0103] "Environmental libraries" are generated from environmental
samples and represent the collective genomes of naturally occurring
organisms archived in cloning vectors that can be propagated in
suitable prokaryotic hosts. Because the cloned DNA is initially
extracted directly from environmental samples, the libraries are
not limited to the small fraction of prokaryotes that can be grown
in pure culture. Additionally, a normalization of the environmental
DNA present in these samples could allow more equal representation
of the DNA from all of the species present in the original sample.
This can dramatically increase the efficiency of finding
interesting genes from minor constituents of the sample which may
be under-represented by several orders of magnitude compared to the
dominant species.
[0104] For example, gene libraries generated from one or more
uncultivated microorganisms are screened for an activity of
interest. Potential pathways encoding bioactive molecules of
interest are first captured in prokaryotic cells in the form of
gene expression libraries. Polynucleotides encoding activities of
interest are isolated from such libraries and introduced into a
host cell. The host cell is grown under conditions which promote
recombination and/or reductive reassortment creating potentially
active biomolecules with novel or enhanced activities.
[0105] The microorganisms from which the polynucleotide may be
prepared include prokaryotic microorganisms, such as Eubacteria and
Archaebacteria, and lower eukaryotic microorganisms such as fungi,
some algae and protozoa. Polynucleotides may be isolated from
environmental samples in which case the nucleic acid may be
recovered without culturing of an organism or recovered from one or
more cultured organisms. In one aspect, such microorganisms may be
extremophiles, such as hyperthermophiles, psychrophiles,
psychrotrophs, halophiles, barophiles and acidophiles.
Polynucleotides encoding enzymes isolated from extremophilic
microorganisms are particularly preferred. Such enzymes may
function at temperatures above 100.degree. C. in terrestrial hot
springs and deep sea thermal vents, at temperatures below 0.degree.
C. in arctic waters, in the saturated salt environment of the Dead
Sea, at pH values around 0 in coal deposits and geothermal
sulfur-rich springs, or at pH values greater than 11 in sewage
sludge. For example, several esterases and lipases cloned and
expressed from extremophilic organisms show high activity
throughout a wide range of temperatures and pHs.
[0106] Polynucleotides selected and isolated as hereinabove
described are introduced into a suitable host cell. A suitable host
cell is any cell which is capable of promoting recombination and/or
reductive reassortment. The selected polynucleotides are preferably
already in a vector which includes appropriate control sequences.
The host cell can be a higher eukaryotic cell, such as a mammalian
cell, or a lower eukaryotic cell, such as a yeast cell, or
preferably, the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the construct into the host cell
can be effected by calcium phosphate transfection, DEAE-Dextran
mediated transfection, or electroporation (Davis et al., 1986).
[0107] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,
COS or Bowes melanoma; adenoviruses; and plant cells. The selection
of an appropriate host is deemed to be within the scope of those
skilled in the art from the teachings herein.
[0108] With particular references to various mammalian cell culture
systems that can be employed to express recombinant protein,
examples of mammalian expression systems include the COS-7 lines of
monkey kidney fibroblasts, described in "SV40-transformed simian
cells support the replication of early SV40 mutants" (Gluzman,
1981), and other cell lines capable of expressing a compatible
vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
Mammalian expression vectors will comprise an origin of
replication, a suitable promoter and enhancer, and also any
necessary ribosome binding sites, polyadenylation site, splice
donor and acceptor sites, transcriptional termination sequences,
and 5' flanking nontranscribed sequences. DNA sequences derived
from the SV40 splice, and polyadenylation sites may be used to
provide the required nontranscribed genetic elements.
[0109] Host cells containing the polynucleotides of interest can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying genes.
The culture conditions, such as temperature, pH and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan. The clones
which are identified as having the specified enzyme activity may
then be sequenced to identify the polynucleotide sequence encoding
an enzyme having the enhanced activity.
[0110] In another aspect, it is envisioned the method of the
present invention can be used to generate novel polynucleotides
encoding biochemical pathways from one or more operons or gene
clusters or portions thereof. For example, bacteria and many
eukaryotes have a coordinated mechanism for regulating genes whose
products are involved in related processes. The genes are
clustered, in structures referred to as "gene clusters," on a
single chromosome and are transcribed together under the control of
a single regulatory sequence, including a single promoter which
initiates transcription of the entire cluster. Thus, a gene cluster
is a group of adjacent genes that are either identical or related,
usually as to their function. An example of a biochemical pathway
encoded by gene clusters are polyketides. Polyketides are molecules
which are an extremely rich source of bioactivities, including
antibiotics (such as tetracyclines and erythromycin), anti-cancer
agents (daunomycin), immunosuppressants (FK506 and rapamycin), and
veterinary products (monensin). Many polyketides (produced by
polyketide synthases) are valuable as therapeutic agents.
Polyketide synthases are multifunctional enzymes that catalyze the
biosynthesis of an enormous variety of carbon chains differing in
length and patterns of functionality and cyclization. Polyketide
synthase genes fall into gene clusters and at least one type
(designated type I) of polyketide synthases have large size genes
and enzymes, complicating genetic manipulation and in vitro studies
of these genes/proteins.
[0111] Gene cluster DNA can be isolated from different organisms
and ligated into vectors, particularly vectors containing
expression regulatory sequences which can control and regulate the
production of a detectable protein or protein-related array
activity from the ligated gene clusters. Use of vectors which have
an exceptionally large capacity for exogenous DNA introduction are
particularly appropriate for use with such gene clusters and are
described by way of example herein to include the f-factor (or
fertility factor) of E. coli. This f-factor of E. coli is a plasmid
which affect high-frequency transfer of itself during conjugation
and is ideal to achieve and stably propagate large DNA fragments,
such as gene clusters from mixed microbial samples. A particularly
preferred embodiment is to use cloning vectors, referred to as
"fosmids" or bacterial artificial chromosome (BAC) vectors. These
are derived from E. coli f-factor which is able to stably integrate
large segments of genomic DNA. When integrated with DNA from a
mixed uncultured environmental sample, this makes it possible to
achieve large genomic fragments in the form of a stable
"environmental DNA library." Another type of vector for use in the
present invention is a cosmid vector. Cosmid vectors were
originally designed to clone and propagate large segments of
genomic DNA. Cloning into cosmid vectors is described in detail in
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,
Cold Spring Harbor Laboratory Press (1989). Once ligated into an
appropriate vector, two or more vectors containing different
polyketide synthase gene clusters can be introduced into a suitable
host cell. Regions of partial sequence homology shared by the gene
clusters will promote processes which result in sequence
reorganization resulting in a hybrid gene cluster. The novel hybrid
gene cluster can then be screened for enhanced activities not found
in the original gene clusters.
[0112] Therefore, in a one embodiment, the invention relates to a
method for producing a biologically active hybrid polypeptide and
screening such a polypeptide for enhanced activity by:
[0113] 1) introducing at least a first polynucleotide in operable
linkage and a second polynucleotide in operable linkage, said at
least first polynucleotide and second polynucleotide sharing at
least one region of partial sequence homology, into a suitable host
cell;
[0114] 2) growing the host cell under conditions which promote
sequence reorganization resulting in a hybrid polynucleotide in
operable linkage;
[0115] 3) expressing a hybrid polypeptide encoded by the hybrid
polynucleotide;
[0116] 4) screening the hybrid polypeptide under conditions which
promote identification of enhanced biological activity; and
[0117] 5) isolating the a polynucleotide encoding the hybrid
polypeptide.
[0118] Methods for screening for various enzyme activities are
known to those of skill in the art and are discussed throughout the
present specification. Such methods may be employed when isolating
the polypeptides and polynucleotides of the invention.
[0119] As representative examples of expression vectors which may
be used, there may be mentioned viral particles, baculovirus,
phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial
chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus,
pseudorabies and derivatives of SV40), P1-based artificial
chromosomes, yeast plasmids, yeast artificial chromosomes, and any
other vectors specific for specific hosts of interest (such as
bacillus, aspergillus and yeast). Thus, for example, the DNA may be
included in any one of a variety of expression vectors for
expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences. Large numbers of
suitable vectors are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example; Bacterial: pQE vectors (Qiagen), pBluescript plasmids,
pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3,
pDR540, pRIT2T (Pharmacia); Eukaryotic: pXTl, pSG5 (Stratagene),
pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid
or other vector may be used so long as they are replicable and
viable in the host. Low copy number or high copy number vectors may
be employed with the present invention.
[0120] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct RNA synthesis. Particular named bacterial promoters
include lacI, lacZ, T3, T7, gpt, lambda P.sub.R, P.sub.L and trp.
Eukaryotic promoters include CMV immediate early, HSV thymidine
kinase, early and late SV40, LTRs from retrovirus, and mouse
metallothionein-I. Selection of the appropriate vector and promoter
is well within the level of ordinary skill in the art. The
expression vector also contains a ribosome binding site for
translation initiation and a transcription terminator. The vector
may also include appropriate sequences for amplifying expression.
Promoter regions can be selected from any desired gene using
chloramphenicol transferase (CAT) vectors or other vectors with
selectable markers. In addition, the expression vectors preferably
contain one or more selectable marker genes to provide a phenotypic
trait for selection of transformed host cells such as dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture, or
such as tetracycline or ampicillin resistance in E. coli.
[0121] In vivo reassortment is focused on "inter-molecular"
processes collectively referred to as "recombination" which in
bacteria, is generally viewed as a "RecA-dependent" phenomenon. The
invention can rely on recombination processes of a host cell to
recombine and re-assort sequences, or the cells' ability to mediate
reductive processes to decrease the complexity of quasi-repeated
sequences in the cell by deletion. This process of "reductive
reassortment" occurs by an "intra-molecular", RecA-independent
process.
[0122] Therefore, in another aspect of the invention, novel
polynucleotides can be generated by the process of reductive
reassortment. The method involves the generation of constructs
containing consecutive sequences (original encoding sequences),
their insertion into an appropriate vector, and their subsequent
introduction into an appropriate host cell. The reassortment of the
individual molecular identities occurs by combinatorial processes
between the consecutive sequences in the construct possessing
regions of homology, or between quasi-repeated units. The
reassortment process recombines and/or reduces the complexity and
extent of the repeated sequences, and results in the production of
novel molecular species. Various treatments may be applied to
enhance the rate of reassorttnent. These could include treatment
with ultra-violet light, or DNA damaging chemicals, and/or the use
of host cell lines displaying enhanced levels of "genetic
instability". Thus the reassortment process may involve homologous
recombination or the natural property of quasi-repeated sequences
to direct their own evolution.
[0123] Repeated or "quasi-repeated" sequences play a role in
genetic instability. In the present invention, "quasi-repeats" are
repeats that are not restricted to their original unit structure.
Quasi-repeated units can be presented as an array of sequences in a
construct; consecutive units of similar sequences. Once ligated,
the junctions between the consecutive sequences become essentially
invisible and the quasi-repetitive nature of the resulting
construct is now continuous at the molecular level. The deletion
process the cell performs to reduce the complexity of the resulting
construct operates between the quasi-repeated sequences. The
quasi-repeated units provide a practically limitless repertoire of
templates upon which slippage events can occur. The constructs
containing the quasi-repeats thus effectively provide sufficient
molecular elasticity that deletion (and potentially insertion)
events can occur virtually anywhere within the quasi-repetitive
units.
[0124] When the quasi-repeated sequences are all ligated in the
same orientation, for instance head to tail or vice versa, the cell
cannot distinguish individual units. Consequently, the reductive
process can occur throughout the sequences. In contrast, when for
example, the units are presented head to head, rather than head to
tail, the inversion delineates the endpoints of the adjacent unit
so that deletion formation will favor the loss of discrete units.
Thus, it is preferable with the present method that the sequences
are in the same orientation. Random orientation of quasi-repeated
sequences will result in the loss of reassortinent efficiency,
while consistent orientation of the sequences will offer the
highest efficiency. However, while having fewer of the contiguous
sequences in the same orientation decreases the efficiency, it may
still provide sufficient elasticity for the effective recovery of
novel molecules. Constructs can be made with the quasi-repeated
sequences in the same orientation to allow higher efficiency.
[0125] Sequences can be assembled in a head to tail orientation
using any of a variety of methods, including the following:
[0126] a) Primers that include a poly-A head and poly-T tail which
when made single-stranded would provide orientation can be
utilized. This is accomplished by having the first few bases of the
primers made from RNA and hence easily removed RNAseH.
[0127] b) Primers that include unique restriction cleavage sites
can be utilized. Multiple sites, a battery of unique sequences, and
repeated synthesis and ligation steps would be required.
[0128] c) The inner few bases of the primer could be thiolated and
an exonuclease used to produce properly tailed molecules.
[0129] The recovery of the re-assorted sequences relies on the
identification of cloning vectors with a reduced repetitive index
(RI). The re-assorted encoding sequences can then be recovered by
amplification. The products are re-cloned and expressed. The
recovery of cloning vectors with reduced RI can be affected by:
[0130] 1) The use of vectors only stably maintained when the
construct is reduced in complexity.
[0131] 2) The physical recovery of shortened vectors by physical
procedures. In this case, the cloning vector would be recovered
using standard plasmid isolation procedures and size fractionated
on either an agarose gel, or column with a low molecular weight cut
off utilizing standard procedures.
[0132] 3) The recovery of vectors containing interrupted genes
which can be selected when insert size decreases.
[0133] 4) The use of direct selection techniques with an expression
vector and the appropriate selection.
[0134] Encoding sequences (for example, genes) from related
organisms may demonstrate a high degree of homology and encode
quite diverse protein products. These types of sequences are
particularly useful in the present invention as quasi-repeats.
However, while the examples illustrated below demonstrate the
reassortment of nearly identical original encoding sequences
(quasi-repeats), this process is not limited to such nearly
identical repeats.
[0135] The following example demonstrates a method of the
invention. Encoding nucleic acid sequences (quasi-repeats) derived
from three (3) unique species are described. Each sequence encodes
a protein with a distinct set of properties. Each of the sequences
differs by a single or a few base pairs at a unique position in the
sequence. The quasi-repeated sequences are separately or
collectively amplified and ligated into random assemblies such that
all possible permutations and combinations are available in the
population of ligated molecules. The number of quasi-repeat units
can be controlled by the assembly conditions. The average number of
quasi-repeated units in a construct is defined as the repetitive
index (RI).
[0136] Once formed, the constructs may, or may not be size
fractionated on an agarose gel according to published protocols,
inserted into a cloning vector, and transfected into an appropriate
host cell. The cells are then propagated and "reductive
reassortment" is effected. The rate of the reductive reassorttnent
process may be stimulated by the introduction of DNA damage if
desired. Whether the reduction in RI is mediated by deletion
formation between repeated sequences by an "intra-molecular"
mechanism, or mediated by recombination-like events through
"inter-molecular" mechanisms is immaterial. The end result is a
reassortment of the molecules into all possible combinations.
[0137] Optionally, the method comprises the additional step of
screening the library members of the shuffled pool to identify
individual shuffled library members having the ability to bind or
otherwise interact, or catalyze a particular reaction (e.g., such
as catalytic domain of an enzyme) with a predetermined
macromolecule, such as for example a proteinaceous receptor, an
oligosaccharide, viron, or other predetermined compound or
structure.
[0138] The polypeptides that are identified from such libraries can
be used for therapeutic, diagnostic, research and related purposes
(e.g., catalysts, solutes for increasing osmolarity of an aqueous
solution, and the like), and/or can be subjected to one or more
additional cycles of shuffling and/or selection.
[0139] In another aspect, it is envisioned that prior to or during
recombination or reassortment, polynucleotides generated by the
method of the invention can be subjected to agents or processes
which promote the introduction of mutations into the original
polynucleotides. The introduction of such mutations would increase
the diversity of resulting hybrid polynucleotides and polypeptides
encoded therefrom. The agents or processes which promote
mutagenesis can include, but are not limited to: (+)-CC-1065, or a
synthetic analog such as (+)-CC-1065-(N3-Adenine (See Sun and
Hurley, (1992); an N-acelylated or deacetylated
4'-fluro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis
(See, for example, van de Poll et al. (1992)); or a N-acetylated or
deacetylated 4-aminobiphenyl adduct capable of inhibiting DNA
synthesis (See also, van de Poll et al. (1992), pp. 751-758);
trivalent chromium, a trivalent chromium salt, a polycyclic
aromatic hydrocarbon (PAH) DNA adduct capable of inhibiting DNA
replication, such as 7-bromomethyl-benz[a]anthracene ("BMA"),
tris(2,3-dibromopropyl)phosphate ("Tris-BP"),
1,2-dibromo-3-chloropropane ("DBCP"), 2-bromoacrolein (2BA),
benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide ("BPDE"), a
platinum(II) halogen salt,
N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline
("N-hydroxy-IQ"), and
N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-p- yridine
("N-hydroxy-PhIP"). Especially preferred means for slowing or
halting PCR amplification consist of UV light (+)-CC-1065 and
(+)-CC-1065-(N3-Adenine). Particularly encompassed means are DNA
adducts or polynucleotides comprising the DNA adducts from the
polynucleotides or polynucleotides pool, which can be released or
removed by a process including heating the solution comprising the
polynucleotides prior to further processing.
[0140] In another aspect the invention is directed to a method of
producing recombinant proteins having biological activity by
treating a sample comprising double-stranded template
polynucleotides encoding a wild-type protein under conditions
according to the invention which provide for the production of
hybrid or re-assorted polynucleotides.
[0141] The invention also provides for the use of proprietary codon
primers (containing a degenerate N,N,N sequence) to introduce point
mutations into a polynucleotide, so as to generate a set of progeny
polypeptides in which a full range of single amino acid
substitutions is represented at each amino acid position (gene site
saturated mutagenesis (GSSM)). The oligos used are comprised
contiguously of a first homologous sequence, a degenerate N,N,N
sequence, and preferably but not necessarily a second homologous
sequence. The downstream progeny translational products from the
use of such oligos include all possible amino acid changes at each
amino acid site along the polypeptide, because the degeneracy of
the N,N,N sequence includes codons for all 20 amino acids.
[0142] In one aspect, one such degenerate oligo (comprised of one
degenerate N,N,N cassette) is used for subjecting each original
codon in a parental polynucleotide template to a full range of
codon substitutions. In another aspect, at least two degenerate
N,N,N cassettes are used--either in the same oligo or not, for
subjecting at least two original codons in a parental
polynucleotide template to a full range of codon substitutions.
Thus, more than one N,N,N sequence can be contained in one oligo to
introduce amino acid mutations at more than one site. This
plurality of N,N,N sequences can be directly contiguous, or
separated by one or more additional nucleotide sequence(s). In
another aspect, oligos serviceable for introducing additions and
deletions can be used either alone or in combination with the
codons containing an N,N,N sequence, to introduce any combination
or permutation of amino acid additions, deletions, and/or
substitutions.
[0143] In a particular exemplification, it is possible to
simultaneously mutagenize two or more contiguous amino acid
positions using an oligo that contains contiguous N,N,N triplets,
i.e. a degenerate (N,N,N).sub.n sequence.
[0144] In another aspect, the present invention provides for the
use of degenerate cassettes having less degeneracy than the N,N,N
sequence. For example, it may be desirable in some instances to use
(e.g. in an oligo) a degenerate triplet sequence comprised of only
one N, where said N can be in the first second or third position of
the triplet. Any other bases including any combinations and
permutations thereof can be used in the remaining two positions of
the triplet. Alternatively, it may be desirable in some instances
to use (e.g., in an oligo) a degenerate N,N,N triplet sequence,
N,N,G/T, or an N,N, G/C triplet sequence.
[0145] It is appreciated, however, that the use of a degenerate
triplet (such as N,N,G/T or an N,N, G/C triplet sequence) as
disclosed in the instant invention is advantageous for several
reasons. In one aspect, this invention provides a means to
systematically and fairly easily generate the substitution of the
full range of possible amino acids (for a total of 20 amino acids)
into each and every amino acid position in a polypeptide. Thus, for
a 100 amino acid polypeptide, the invention provides a way to
systematically and fairly easily generate 2000 distinct species
(i.e., 20 possible amino acids per position times 100 amino acid
positions). It is appreciated that there is provided, through the
use of an oligo containing a degenerate N,N,G/T or an N,N, G/C
triplet sequence, 32 individual sequences that code for 20 possible
amino acids. Thus, in a reaction vessel in which a parental
polynucleotide sequence is subjected to saturation mutagenesis
using one such oligo, there are generated 32 distinct progeny
polynucleotides encoding 20 distinct polypeptides. In contrast, the
use of a non-degenerate oligo in site-directed mutagenesis leads to
only one progeny polypeptide product per reaction vessel.
[0146] This invention also provides for the use of nondegenerate
oligos, which can optionally be used in combination with degenerate
primers disclosed. It is appreciated that in some situations, it is
advantageous to use nondegenerate oligos to generate specific point
mutations in a working polynucleotide. This provides a means to
generate specific silent point mutations, point mutations leading
to corresponding amino acid changes, and point mutations that cause
the generation of stop codons and the corresponding expression of
polypeptide fragments.
[0147] Thus, in a preferred embodiment of this invention, each
saturation mutagenesis reaction vessel contains polynucleotides
encoding at least 20 progeny polypeptide molecules such that all 20
amino acids are represented at the one specific amino acid position
corresponding to the codon position mutagenized in the parental
polynucleotide. The 32-fold degenerate progeny polypeptides
generated from each saturation mutagenesis reaction vessel can be
subjected to clonal amplification (e.g., cloned into a suitable E.
coli host using an expression vector) and subjected to expression
screening. When an individual progeny polypeptide is identified by
screening to display a favorable change in property (when compared
to the parental polypeptide), it can be sequenced to identify the
correspondingly favorable amino acid substitution contained
therein.
[0148] It is appreciated that upon mutagenizing each and every
amino acid position in a parental polypeptide using saturation
mutagenesis as disclosed herein, favorable amino acid changes may
be identified at more than one amino acid position. One or more new
progeny molecules can be generated that contain a combination of
all or part of these favorable amino acid substitutions. For
example, if 2 specific favorable amino acid changes are identified
in each of 3 amino acid positions in a polypeptide, the
permutations include 3 possibilities at each position (no change
from the original amino acid, and each of two favorable changes)
and 3 positions. Thus, there are 3.times.3.times.3 or 27 total
possibilities, including 7 that were previously examined--6 single
point mutations (i.e., 2 at each of three positions) and no change
at any position.
[0149] In yet another aspect, site-saturation mutagenesis can be
used together with shuffling, chimerization, recombination and
other mutagenizing processes, along with screening. This invention
provides for the use of any mutagenizing process(es), including
saturation mutagenesis, in an iterative manner. In one
exemplification, the iterative use of any mutagenizing process(es)
is used in combination with screening.
[0150] Thus, in a non-limiting exemplification, this invention
provides for the use of saturation mutagenesis in combination with
additional mutagenization processes, such as process where two or
more related polynucleotides are introduced into a suitable host
cell such that a hybrid polynucleotide is generated by
recombination and reductive reassortment.
[0151] In addition to performing mutagenesis along the entire
sequence of a gene, the instant invention provides that mutagenesis
can be use to replace each of any number of bases in a
polynucleotide sequence, wherein the number of bases to be
mutagenized is preferably every integer from 15 to 100,000. Thus,
instead of mutagenizing every position along a molecule, one can
subject every or a discrete number of bases (preferably a subset
totaling from 15 to 100,000) to mutagenesis. Preferably, a separate
nucleotide is used for mutagenizing each position or group of
positions along a polynucleotide sequence. A group of 3 positions
to be mutagenized may be a codon. The mutations are preferably
introduced using a mutagenic primer, containing a heterologous
cassette, also referred to as a mutagenic cassette. Preferred
cassettes can have from 1 to 500 bases. Each nucleotide position in
such heterologous cassettes be N, A, C, G, T, A/C, A/G, A/T, C/G,
C/T, G/T, C/G/T, A/G/T, A/C/T, A/C/G, or E, where E is any base
that is not A, C, G, or T (E can be referred to as a designer
oligo).
[0152] In a general sense, saturation mutagenesis is comprised of
mutagenizing a complete set of mutagenic cassettes (wherein each
cassette is preferably about 1-500 bases in length) in defined
polynucleotide sequence to be mutagenized (wherein the sequence to
be mutagenized is preferably from about 15 to 100,000 bases in
length). Thus, a group of mutations (ranging from 1 to 100
mutations) is introduced into each cassette to be mutagenized. A
grouping of mutations to be introduced into one cassette can be
different or the same from a second grouping of mutations to be
introduced into a second cassette during the application of one
round of saturation mutagenesis. Such groupings are exemplified by
deletions, additions, groupings of particular codons, and groupings
of particular nucleotide cassettes.
[0153] Defined sequences to be mutagenized include a whole gene,
pathway, cDNA, an entire open reading frame (ORF), and entire
promoter, enhancer, repressor/transactivator, origin of
replication, intron, operator, or any polynucleotide functional
group. Generally, a "defined sequences" for this purpose may be any
polynucleotide that a 15 base-polynucleotide sequence, and
polynucleotide sequences of lengths between 15 bases and 15,000
bases (this invention specifically names every integer in between).
Considerations in choosing groupings of codons include types of
amino acids encoded by a degenerate mutagenic cassette.
[0154] In a particularly preferred exemplification a grouping of
mutations that can be introduced into a mutagenic cassette, this
invention specifically provides for degenerate codon substitutions
(using degenerate oligos) that code for 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids at each
position, and a library of polypeptides encoded thereby.
[0155] One aspect of the invention is an isolated nucleic acid
comprising one of the sequences of Group A nucleic acid sequences,
and sequences substantially identical thereto, the sequences
complementary thereto, or a fragment comprising at least 10, 15,
20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500
consecutive bases of one of the sequences of a Group A nucleic acid
sequence (or the sequences complementary thereto). The isolated,
nucleic acids may comprise DNA, including cDNA, genomic DNA, and
synthetic DNA. The DNA may be double-stranded or single-stranded,
and if single stranded may be the coding strand or non-coding
(anti-sense) strand. Alternatively, the isolated nucleic acids may
comprise RNA.
[0156] As discussed in more detail below, the isolated nucleic
acids of one of the Group A nucleic acid sequences, and sequences
substantially identical thereto, may be used to prepare one of the
polypeptides of a Group B amino acid sequence, and sequences
substantially identical thereto, or fragments comprising at least
5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive
amino acids of one of the polypeptides of Group B amino acid
sequences, and sequences substantially identical thereto.
[0157] Accordingly, another aspect of the invention is an isolated
nucleic acid which encodes one of the polypeptides of Group B amino
acid sequences, and sequences substantially identical thereto, or
fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50,
75, 100, or 150 consecutive amino acids of one of the polypeptides
of the Group B amino acid sequences. The coding sequences of these
nucleic acids may be identical to one of the coding sequences of
one of the nucleic acids of Group A nucleic acid sequences, or a
fragment thereof or may be different coding sequences which encode
one of the polypeptides of Group B amino acid sequences, sequences
substantially identical thereto, and fragments having at least 5,
10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino
acids of one of the polypeptides of Group B amino acid sequences,
as a result of the redundancy or degeneracy of the genetic code.
The genetic code is well known to those of skill in the art and can
be obtained, for example, on page 214 of B. Lewin, Genes VI, Oxford
University Press, 1997, the disclosure of which is incorporated
herein by reference.
[0158] The isolated nucleic acid which encodes one of the
polypeptides of Group B amino acid sequences, and sequences
substantially identical thereto, may include, but is not limited
to: only the coding sequence of one of Group A nucleic acid
sequences, and sequences substantially identical thereto, and
additional coding sequences, such as leader sequences or proprotein
sequences and non-coding sequences, such as introns or non-coding
sequences 5' and/or 3' of the coding sequence. Thus, as used
herein, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only the coding
sequence for the polypeptide as well as a polynucleotide which
includes additional coding and/or non-coding sequence.
[0159] Alternatively, the nucleic acid sequences of Group A nucleic
acid sequences, and sequences substantially identical thereto, may
be mutagenized using conventional techniques, such as site directed
mutagenesis, or other techniques familiar to those skilled in the
art, to introduce silent changes into the polynucleotides of Group
A nucleic acid sequences, and sequences substantially identical
thereto. As used herein, "silent changes" include, for example,
changes which do not alter the amino acid sequence encoded by the
polynucleotide. Such changes may be desirable in order to increase
the level of the polypeptide produced by host cells containing a
vector encoding the polypeptide by introducing codons or codon
pairs which occur frequently in the host organism.
[0160] The invention also relates to polynucleotides which have
nucleotide changes which result in amino acid substitutions,
additions, deletions, fusions and truncations in the polypeptides
of Group B amino acid sequences, and sequences substantially
identical thereto. Such nucleotide changes may be introduced using
techniques such as site directed mutagenesis, random chemical
mutagenesis, exonuclease III deletion, and other recombinant DNA
techniques. Alternatively, such nucleotide changes may be naturally
occurring allelic variants which are isolated by identifying
nucleic acids which specifically hybridize to probes comprising at
least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400,
or 500 consecutive bases of one of the sequences of Group A nucleic
acid sequences, and sequences substantially identical thereto (or
the sequences complementary thereto) under conditions of high,
moderate, or low stringency as provided herein.
[0161] The isolated nucleic acids of Group A nucleic acid
sequences, and sequences substantially identical thereto, the
sequences complementary thereto, or a fragment comprising at least
10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500
consecutive bases of one of the sequences of Group A nucleic acid
sequences, and sequences substantially identical thereto, or the
sequences complementary thereto may also be used as probes to
determine whether a biological sample, such as a soil sample,
contains an organism having a nucleic acid sequence of the
invention or an organism from which the nucleic acid was obtained.
In such procedures, a biological sample potentially harboring the
organism from which the nucleic acid was isolated is obtained and
nucleic acids are obtained from the sample. The nucleic acids are
contacted with the probe under conditions which permit the probe to
specifically hybridize to any complementary sequences from which
are present therein.
[0162] Where necessary, conditions which permit the probe to
specifically hybridize to complementary sequences may be determined
by placing the probe in contact with complementary sequences from
samples known to contain the complementary sequence as well as
control sequences which do not contain the complementary sequence.
Hybridization conditions, such as the salt concentration of the
hybridization buffer, the formamide concentration of the
hybridization buffer, or the hybridization temperature, may be
varied to identify conditions which allow the probe to hybridize
specifically to complementary nucleic acids.
[0163] If the sample contains the organism from which the nucleic
acid was isolated, specific hybridization of the probe is then
detected. Hybridization may be detected by labeling the probe with
a detectable agent such as a radioactive isotope, a fluorescent dye
or an enzyme capable of catalyzing the formation of a detectable
product.
[0164] Many methods for using the labeled probes to detect the
presence of complementary nucleic acids in a sample are familiar to
those skilled in the art. These include Southern Blots, Northern
Blots, colony hybridization procedures, and dot blots. Protocols
for each of these procedures are provided in Ausubel et al. Current
Protocols in Molecular Biology, John Wiley 503 Sons, Inc. (1997)
and Sambrook et al., Molecular Cloning A Laboratory Manual 2nd Ed.,
Cold Spring Harbor Laboratory Press (1989), the entire disclosures
of which are incorporated herein by reference.
[0165] Alternatively, more than one probe (at least one of which is
capable of specifically hybridizing to any complementary sequences
which are present in the nucleic acid sample), may be used in an
amplification reaction to determine whether the sample contains an
organism containing a nucleic acid sequence of the invention (e.g.,
an organism from which the nucleic acid was isolated). Typically,
the probes comprise oligonucleotides. In one embodiment, the
amplification reaction may comprise a PCR reaction. PCR protocols
are described in Ausubel and Sambrook, supra. Alternatively, the
amplification may comprise a ligase chain reaction, 3SR, or strand
displacement reaction. (See Barany, F., "The Ligase Chain Reaction
in a PCR World", PCR Methods and Applications 1:5-16, 1991; E. Fahy
et al., "Self-sustained Sequence Replication (3SR): An Isothermal
Transcription-based Amplification System Alternative to PCR", PCR
Methods and Applications 1:25-33, 1991; and Walker G. T. et al.,
"Strand Displacement Amplification-an Isothermal in vitro DNA
Amplification Technique", Nucleic Acid Research 20:1691-1696, 1992,
the disclosures of which are incorporated herein by reference in
their entireties). In such procedures, the nucleic acids in the
sample are contacted with the probes, the amplification reaction is
performed, and any resulting amplification product is detected. The
amplification product may be detected by performing gel
electrophoresis on the reaction products and staining the gel with
an interculator such as ethidium bromide. Alternatively, one or
more of the probes may be labeled with a radioactive isotope and
the presence of a radioactive amplification product may be detected
by autoradiography after gel electrophoresis.
[0166] Probes derived from sequences near the ends of the sequences
of Group A nucleic acid sequences, and sequences substantially
identical thereto, may also be used in chromosome walking
procedures to identify clones containing genomic sequences located
adjacent to the sequences of Group A nucleic acid sequences, and
sequences substantially identical thereto. Such methods allow the
isolation of genes which encode additional proteins from the host
organism.
[0167] The isolated nucleic acids of Group A nucleic acid
sequences, and sequences substantially identical thereto, the
sequences complementary thereto, or a fragment comprising at least
10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500
consecutive bases of one of the sequences of Group A nucleic acid
sequences, and sequences substantially identical thereto, or the
sequences complementary thereto may be used as probes to identify
and isolate related nucleic acids. In some embodiments, the related
nucleic acids may be cDNAs or genomic DNAs from organisms other
than the one from which the nucleic acid was isolated. For example,
the other organisms may be related organisms. In such procedures, a
nucleic acid sample is contacted with the probe under conditions
which permit the probe to specifically hybridize to related
sequences. Hybridization of the probe to nucleic acids from the
related organism is then detected using any of the methods
described above.
[0168] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter.
[0169] Hybridization may be carried out under conditions of low
stringency, moderate stringency or high stringency. As an example
of nucleic acid hybridization, a polymer membrane containing
immobilized denatured nucleic acids is first prehybridized for 30
minutes at 45.degree. C. in a solution consisting of 0.9 M NaCl, 50
mM NaH.sub.2PO.sub.4, pH 7.0, 5.0 mM Na.sub.2EDTA, 0.5% SDS,
10.times. Denhardt's, and 0.5 mg/ml polyriboadenylic acid.
Approximately 2.times.10.sup.7 cpm (specific activity
4-9.times.10.sup.8 cpM/ug) of .sup.32P end-labeled oligonucleotide
probe are then added to the solution. After 12-16 hours of
incubation, the membrane is washed for 30 minutes at room
temperature in 1.times. SET (150 mM NaCl, 20 mM Tris hydrochloride,
pH 7.8, 1 mM Na.sub.2EDTA) containing 0.5% SDS, followed by a 30
minute wash in fresh 1.times. SET at T.sub.m-10.degree. C. for the
oligonucleotide probe. The membrane is then exposed to
auto-radiographic film for detection of hybridization signals.
[0170] By varying the stringency of the hybridization conditions
used to identify nucleic acids, such as cDNAs or genomic DNAs,
which hybridize to the detectable probe, nucleic acids having
different levels of homology to the probe can be identified and
isolated. Stringency may be varied by conducting the hybridization
at varying temperatures below the melting temperatures of the
probes. The melting temperature, T.sub.m, is the temperature (under
defined ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly complementary probe. Very stringent
conditions are selected to be equal to or about 5.degree. C. lower
than the T.sub.m for a particular probe. The melting temperature of
the probe may be calculated using the following formulas:
[0171] For probes between 14 and 70 nucleotides in length the
melting temperature (T.sub.m) is calculated using the formula:
T.sub.m=81.5+16.6(log [Na+])+0.41(fraction G+C)-(600/N) where N is
the length of the probe.
[0172] If the hybridization is carried out in a solution containing
formamide, the melting temperature may be calculated using the
equation: T.sub.m=81.5+16.6(log [Na+])+0.41(fraction G+C)-(0.63%
formamide)-(600/N) where N is the length of the probe.
[0173] Prehybridization may be carried out in 6.times.SSC, 5.times.
Denhardt's reagent, 0.5% SDS, 100 .mu.g denatured fragmented salmon
sperm DNA or 6.times.SSC, 5.times. Denhardt's reagent, 0.5% SDS,
100 .mu.g denatured fragmented salmon sperm DNA, 50% formamide. The
formulas for SSC and Denhardt's solutions are listed in Sambrook et
al., supra.
[0174] Hybridization is conducted by adding the detectable probe to
the prehybridization solutions listed above. Where the probe
comprises double stranded DNA, it is denatured before addition to
the hybridization solution. The filter is contacted with the
hybridization solution for a sufficient period of time to allow the
probe to hybridize to cDNAs or genomic DNAs containing sequences
complementary thereto or homologous thereto. For probes over 200
nucleotides in length, the hybridization may be carried out at
15-25.degree. C. below the T.sub.m. For shorter probes, such as
oligonucleotide probes, the hybridization may be conducted at
5-10.degree. C. below the T.sub.m. Typically, for hybridizations in
6.times.SSC, the hybridization is conducted at approximately
68.degree. C. Usually, for hybridizations in 50% formamide
containing solutions, the hybridization is conducted at
approximately 42.degree. C.
[0175] All of the foregoing hybridizations would be considered to
be under conditions of high stringency.
[0176] Following hybridization, the filter is washed to remove any
non-specifically bound detectable probe. The stringency used to
wash the filters can also be varied depending on the nature of the
nucleic acids being hybridized, the length of the nucleic acids
being hybridized, the degree of complementarity, the nucleotide
sequence composition (e.g., GC v. AT content), and the nucleic acid
type (e.g., RNA v. DNA). Examples of progressively higher
stringency condition washes are as follows: 2.times.SSC, 0.1% SDS
at room temperature for 15 minutes (low stringency); 0.1.times.SSC,
0.5% SDS at room temperature for 30 minutes to 1 hour (moderate
stringency); 0.1.times.SSC, 0.5% SDS for 15 to 30 minutes at
between the hybridization temperature and 68.degree. C. (high
stringency); and 0.15M NaCl for 15 minutes at 72.degree. C. (very
high stringency). A final low stringency wash can be conducted in
0.1.times.SSC at room temperature. The examples above are merely
illustrative of one set of conditions that can be used to wash
filters. One of skill in the art would know that there are numerous
recipes for different stringency washes. Some other examples are
given below.
[0177] Nucleic acids which have hybridized to the probe are
identified by autoradiography or other conventional techniques.
[0178] The above procedure may be modified to identify nucleic
acids having decreasing levels of homology to the probe sequence.
For example, to obtain nucleic acids of decreasing homology to the
detectable probe, less stringent conditions may be used. For
example, the hybridization temperature may be decreased in
increments of 5.degree. C. from 68.degree. C. to 42.degree. C. in a
hybridization buffer having a Na+ concentration of approximately
1M. Following hybridization, the filter may be washed with
2.times.SSC, 0.5% SDS at the temperature of hybridization. These
conditions are considered to be "moderate" conditions above
50.degree. C. and "low" conditions below 50.degree. C. A specific
example of "moderate" hybridization conditions is when the above
hybridization is conducted at 55.degree. C. A specific example of
"low stringency" hybridization conditions is when the above
hybridization is conducted at 45.degree. C.
[0179] Alternatively, the hybridization may be carried out in
buffers, such as 6.times.SSC, containing formamide at a temperature
of 42.degree. C. In this case, the concentration of formamide in
the hybridization buffer may be reduced in 5% increments from 50%
to 0% to identify clones having decreasing levels of homology to
the probe. Following hybridization, the filter may be washed with
6.times.SSC, 0.5% SDS at 50.degree. C. These conditions are
considered to be "moderate" conditions above 25% formamide and
"low" conditions below 25% formamide. A specific example of
"moderate" hybridization conditions is when the above hybridization
is conducted at 30% formamide. A specific example of "low
stringency" hybridization conditions is when the above
hybridization is conducted at 10% formamide.
[0180] For example, the preceding methods may be used to isolate
nucleic acids having a sequence with at least about 97%, at least
95%, at least 90%, at least 85%, at least 80%, at least 75%, at
least 70%, at least 65%, at least 60%, at least 55%, or at least
50% homology to a nucleic acid sequence selected from the group
consisting of one of the sequences of Group A nucleic acid
sequences, and sequences substantially identical thereto, or
fragments comprising at least about 10, 15, 20, 25, 30, 35, 40, 50,
75, 100, 150, 200, 300, 400, or 500 consecutive bases thereof, and
the sequences complementary thereto. Homology may be measured using
the alignment algorithm. For example, the homologous
polynucleotides may have a coding sequence which is a naturally
occurring allelic variant of one of the coding sequences described
herein. Such allelic variants may have a substitution, deletion or
addition of one or more nucleotides when compared to the nucleic
acids of Group A nucleic acid sequences or the sequences
complementary thereto.
[0181] Additionally, the above procedures may be used to isolate
nucleic acids which encode polypeptides having at least about 99%,
95%, at least 90%, at least 85%, at least 80%, at least 75%, at
least 70%, at least 65%, at least 60%, at least 55%, or at least
50% homology to a polypeptide having the sequence of one of Group B
amino acid sequences, and sequences substantially identical
thereto, or fragments comprising at least 5, 10, 15, 20, 25, 30,
35, 40, 50, 75, 100, or 150 consecutive amino acids thereof as
determined using a sequence alignment algorithm (e.g., such as the
FASTA version 3.0t78 algorithm with the default parameters).
[0182] Another aspect of the invention is an isolated or purified
polypeptide comprising the sequence of one of Group A nucleic acid
sequences, and sequences substantially identical thereto, or
fragments comprising at least about 5, 10, 15, 20, 25, 30, 35, 40,
50, 75, 100, or 150 consecutive amino acids thereof. As discussed
above, such polypeptides may be obtained by inserting a nucleic
acid encoding the polypeptide into a vector such that the coding
sequence is operably linked to a sequence capable of driving the
expression of the encoded polypeptide in a suitable host cell. For
example, the expression vector may comprise a promoter, a ribosome
binding site for translation initiation and a transcription
terminator. The vector may also include appropriate sequences for
amplifying expression.
[0183] Promoters suitable for expressing the polypeptide or
fragment thereof in bacteria include the E. coli lac or trp
promoters, the lacI promoter, the lacZ promoter, the T3 promoter,
the T7 promoter, the gpt promoter, the lambda P.sub.R promoter, the
lambda P.sub.L promoter, promoters from operons encoding glycolytic
enzymes such as 3-phosphoglycerate kinase (PGK), and the acid
phosphatase promoter. Fungal promoters include the .A-inverted.
factor promoter. Eukaryotic promoters include the CMV immediate
early promoter, the HSV thymidine kinase promoter, heat shock
promoters, the early and late SV40 promoter, LTRs from
retroviruses, and the mouse metallothionein-I promoter. Other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses may also be used.
[0184] Mammalian expression vectors may also comprise an origin of
replication, any necessary ribosome binding sites, a
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. In some embodiments, DNA sequences
derived from the SV40 splice and polyadenylation sites may be used
to provide the required nontranscribed genetic elements.
[0185] Vectors for expressing the polypeptide or fragment thereof
in eukaryotic cells may also contain enhancers to increase
expression levels. Enhancers are cis-acting elements of DNA,
usually from about 10 to about 300 bp in length that act on a
promoter to increase its transcription. Examples include the SV40
enhancer on the late side of the replication origin bp 100 to 270,
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and the adenovirus
enhancers.
[0186] In addition, the expression vectors typically contain one or
more selectable marker genes to permit selection of host cells
containing the vector. Such selectable markers include genes
encoding dihydrofolate reductase or genes conferring neomycin
resistance for eukaryotic cell culture, genes conferring
tetracycline or ampicillin resistance in E. coli, and the S.
cerevisiae TRP1 gene.
[0187] In some embodiments, the nucleic acid encoding one of the
polypeptides of Group B amino acid sequences, and sequences
substantially identical thereto, or fragments comprising at least
about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids thereof is assembled in appropriate phase
with a leader sequence capable of directing secretion of the
translated polypeptide or fragment thereof. Optionally, the nucleic
acid can encode a fusion polypeptide in which one of the
polypeptides of Group B amino acid sequences, and sequences
substantially identical thereto, or fragments comprising at least
5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive
amino acids thereof is fused to heterologous peptides or
polypeptides, such as N-terminal identification peptides which
impart desired characteristics, such as increased stability or
simplified purification.
[0188] The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA sequence is ligated
to the desired position in the vector following digestion of the
insert and the vector with appropriate restriction endonucleases.
Alternatively, blunt ends in both the insert and the vector may be
ligated. A variety of cloning techniques are disclosed in Ausubel
et al. Current Protocols in Molecular Biology, John Wiley 503 Sons,
Inc. 1997 and Sambrook et al., Molecular Cloning: A Laboratory
Manual 2nd Ed., Cold Spring Harbor Laboratory Press (1989), the
entire disclosures of which are incorporated herein by reference.
Such procedures and others are deemed to be within the scope of
those skilled in the art.
[0189] The vector may be, for example, in the form of a plasmid, a
viral particle, or a phage. Other vectors include chromosomal,
nonchromosomal and synthetic DNA sequences, derivatives of SV40;
bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors
derived from combinations of plasmids and phage DNA, viral DNA such
as vaccinia, adenovirus, fowl pox virus, and pseudorabies. A
variety of cloning and expression vectors for use with prokaryotic
and eukaryofic hosts are described by Sambrook, et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.,
(1989), the disclosure of which is hereby incorporated by
reference.
[0190] Particular bacterial vectors which may be used include the
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia
Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison,
Wis., USA) pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174 pBluescript
II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a,
pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8 and pCM7.
Particular eukaryotic vectors include pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharnacia). However, any
other vector may be used as long as it is replicable and viable in
the host cell.
[0191] The host cell may be any of the host cells familiar to those
skilled in the art, including prokaryotic cells, eukaryotic cells,
mammalian cells, insect cells, or plant cells. As representative
examples of appropriate hosts, there may be mentioned: bacterial
cells, such as E. coli, Streptomyces, Bacillus subtilis, Salmonella
typhimurium and various species within the genera Pseudomonas,
Streptomyces, and Staphylococcus, fungal cells, such as yeast,
insect cells such as Drosophila S2 and Spodoptera Sf9, animal cells
such as CHO, COS or Bowes melanoma, and adenoviruses. The selection
of an appropriate host is within the abilities of those skilled in
the art.
[0192] The vector may be introduced into the host cells using any
of a variety of techniques, including transformation, transfection,
transduction, viral infection, gene guns, or Ti-mediated gene
transfer. Particular methods include calcium phosphate
transfection, DEAE-Dextran mediated transfection, lipofection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0193] Where appropriate, the engineered host cells can be cultured
in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
genes of the invention. Following transformation of a suitable host
strain and growth of the host strain to an appropriate cell
density, the selected promoter may be induced by appropriate means
(e.g., temperature shift or chemical induction) and the cells may
be cultured for an additional period to allow them to produce the
desired polypeptide or fragment thereof.
[0194] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract is
retained for further purification. Microbial cells employed for
expression of proteins can be disrupted by any convenient method,
including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents. Such methods are well known to those
skilled in the art. The expressed polypeptide or fragment thereof
can be recovered and purified from recombinant cell cultures by
methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Protein refolding steps
can be used, as necessary, in completing configuration of the
polypeptide. If desired, high performance liquid chromatography
(HPLC) can be employed for final purification steps.
[0195] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts
(described by Gluzman, Cell, 23:175, 1981), and other cell lines
capable of expressing proteins from a compatible vector, such as
the C127, 3T3, CHO, HeLa and BHK cell lines.
[0196] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Depending upon the host employed in a recombinant
production procedure, the polypeptides produced by host cells
containing the vector may be glycosylated or may be
non-glycosylated. Polypeptides of the invention may or may not also
include an initial methionine amino acid residue.
[0197] Alternatively, the polypeptides of Group B amino acid
sequences, and sequences substantially identical thereto, or
fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50,
75, 100, or 150 consecutive amino acids thereof can be
synthetically produced by conventional peptide synthesizers. In
other embodiments, fragments or portions of the polypeptides may be
employed for producing the corresponding full-length polypeptide by
peptide synthesis; therefore, the fragments may be employed as
intermediates for producing the full-length polypeptides.
[0198] Cell-free translation systems can also be employed to
produce one of the polypeptides of Group B amino acid sequences,
and sequences substantially identical thereto, or fragments
comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or
150 consecutive amino acids thereof using mRNAs transcribed from a
DNA construct comprising a promoter operably linked to a nucleic
acid encoding the polypeptide or fragment thereof. In some
embodiments, the DNA construct may be linearized prior to
conducting an in vitro transcription reaction. The transcribed mRNA
is then incubated with an appropriate cell-free translation
extract, such as a rabbit reticulocyte extract, to produce the
desired polypeptide or fragment thereof.
[0199] The invention also relates to variants of the polypeptides
of Group B amino acid sequences, and sequences substantially
identical thereto, or fragments comprising at least 5, 10, 15, 20,
25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids
thereof. The term "variant" includes derivatives or analogs of
these polypeptides. In particular, the variants may differ in amino
acid sequence from the polypeptides of Group B amino acid
sequences, and sequences substantially identical thereto, by one or
more substitutions, additions, deletions, fusions and truncations,
which may be present in any combination.
[0200] The variants may be naturally occurring or created in vitro.
In particular, such variants may be created using genetic
engineering techniques such as site directed mutagenesis, random
chemical mutagenesis, Exonuclease III deletion procedures, and
standard cloning techniques. Alternatively, such variants,
fragments, analogs, or derivatives may be created using chemical
synthesis or modification procedures.
[0201] Other methods of making variants are also familiar to those
skilled in the art. These include procedures in which nucleic acid
sequences obtained from natural isolates are modified to generate
nucleic acids which encode polypeptides having characteristics
which enhance their value in industrial or laboratory applications.
In such procedures, a large number of variant sequences having one
or more nucleotide differences with respect to the sequence
obtained from the natural isolate are generated and characterized.
Typically, these nucleotide differences result in amino acid
changes with respect to the polypeptides encoded by the nucleic
acids from the natural isolates.
[0202] For example, variants may be created using error prone PCR.
In error prone PCR, PCR is performed under conditions where the
copying fidelity of the DNA polymerase is low, such that a high
rate of point mutations is obtained along the entire length of the
PCR product. Error prone PCR is described in Leung, D. W., et al.,
Technique, 1:11-15, 1989) and Caldwell, R. C. & Joyce G. F.,
PCR Methods Applic., 2:28-33, 1992, the disclosure of which is
incorporated herein by reference in its entirety. Briefly, in such
procedures, nucleic acids to be mutagenized are mixed with PCR
primers, reaction buffer, MgCl.sub.2, MnCl.sub.2, Taq polymerase
and an appropriate concentration of dNTPs for achieving a high rate
of point mutation along the entire length of the PCR product. For
example, the reaction may be performed using 20 fmoles of nucleic
acid to be mutagenized, 30 pmole of each PCR primer, a reaction
buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01%
gelatin, 7 mM MgCl.sub.2, 0.5 mM MnCl.sub.2, 5 units of Taq
polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR
may be performed for 30 cycles of 94.degree. C. for 1 min,
45.degree. C. for 1 min, and 72.degree. C. for 1 min. However, it
will be appreciated that these parameters may be varied as
appropriate. The mutagenized nucleic acids are cloned into an
appropriate vector and the activities of the polypeptides encoded
by the mutagenized nucleic acids is evaluated.
[0203] Variants may also be created using oligonucleotide directed
mutagenesis to generate site-specific mutations in any cloned DNA
of interest. Oligonucleotide mutagenesis is described in
Reidhaar-Olson, J. F. & Sauer, R. T., et al., Science,
241:53-57, 1988, the disclosure of which is incorporated herein by
reference in its entirety. Briefly, in such procedures a plurality
of double stranded oligonucleotides bearing one or more mutations
to be introduced into the cloned DNA are synthesized and inserted
into the cloned DNA to be mutagenized. Clones containing the
mutagenized DNA are recovered and the activities of the
polypeptides they encode are assessed.
[0204] Another method for generating variants is assembly PCR.
Assembly PCR involves the assembly of a PCR product from a mixture
of small DNA fragments. A large number of different PCR reactions
occur in parallel in the same vial, with the products of one
reaction priming the products of another reaction. Assembly PCR is
described in U.S. Pat. No. 5,965,408, filed Jul. 9, 1996, entitled,
"Method of DNA Reassembly by Interrupting Synthesis", the
disclosure of which is incorporated herein by reference in its
entirety.
[0205] Still another method of generating variants is sexual PCR
mutagenesis. In sexual PCR mutagenesis, forced homologous
recombination occurs between DNA molecules of different but highly
related DNA sequence in vitro, as a result of random fragmentation
of the DNA molecule based on sequence homology, followed by
fixation of the crossover by primer extension in a PCR reaction.
Sexual PCR mutagenesis is described in Stemmer, W. P., PNAS, USA,
91:10747-10751, 1994, the disclosure of which is incorporated
herein by reference. Briefly, in such procedures a plurality of
nucleic acids to be recombined are digested with DNAse to generate
fragments having an average size of 50-200 nucleotides. Fragments
of the desired average size are purified and resuspended in a PCR
mixture. PCR is conducted under conditions which facilitate
recombination between the nucleic acid fragments. For example, PCR
may be performed by resuspending the purified fragments at a
concentration of 10-30 ng/:l in a solution of 0.2 mM of each dNTP,
2.2 mM MgCl2, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton
X-100. 2.5 units of Taq polymerase per 100:1 of reaction mixture is
added and PCR is performed using the following regime: 94.degree.
C. for 60 seconds, 94.degree. C. for 30 seconds, 50-55.degree. C.
for 30 seconds, 72.degree. C. for 30 seconds (30-45 times) and
72.degree. C. for 5 minutes. However, it will be appreciated that
these parameters may be varied as appropriate. In some embodiments,
oligonucleotides may be included in the PCR reactions. In other
embodiments, the Klenow fragment of DNA polymerase I may be used in
a first set of PCR reactions and Taq polymerase may be used in a
subsequent set of PCR reactions. Recombinant sequences are isolated
and the activities of the polypeptides they encode are
assessed.
[0206] Variants may also be created by in vivo mutagenesis. In some
embodiments, random mutations in a sequence of interest are
generated by propagating the sequence of interest in a bacterial
strain, such as an E. coli strain, which carries mutations in one
or more of the DNA repair pathways. Such "mutator" strains have a
higher random mutation rate than that of a wild-type parent.
Propagating the DNA in one of these strains will eventually
generate random mutations within the DNA. Mutator strains suitable
for use for in vivo mutagenesis are described in PCT Publication
No. WO 91/16427, published Oct. 31, 1991, entitled "Methods for
Phenotype Creation from Multiple Gene Populations" the disclosure
of which is incorporated herein by reference in its entirety.
[0207] Variants may also be generated using cassette mutagenesis.
In cassette mutagenesis a small region of a double stranded DNA
molecule is replaced with a synthetic oligonucleotide "cassette"
that differs from the native sequence. The oligonucleotide often
contains completely and/or partially randomized native
sequence.
[0208] Recursive ensemble mutagenesis may also be used to generate
variants. Recursive ensemble mutagenesis is an algorithm for
protein engineering (protein mutagenesis) developed to produce
diverse populations of phenotypically related mutants whose members
differ in amino acid sequence. This method uses a feedback
mechanism to control successive rounds of combinatorial cassette
mutagenesis. Recursive ensemble mutagenesis is described in Arkin,
A. P. and Youvan, D. C., PNAS, USA, 89:7811-7815, 1992, the
disclosure of which is incorporated herein by reference in its
entirety.
[0209] In some embodiments, variants are created using exponential
ensemble mutagenesis. Exponential ensemble mutagenesis is a process
for generating combinatorial libraries with a high percentage of
unique and functional mutants, wherein small groups of residues are
randomized in parallel to identify, at each altered position, amino
acids which lead to functional proteins. Exponential ensemble
mutagenesis is described in Delegrave, S. and Youvan, D. C.,
Biotechnology Research, 11:1548-1552, 1993, the disclosure of which
incorporated herein by reference in its entirety. Random and
site-directed mutagenesis are described in Arnold, F. H., Current
Opinion in Biotechnology, 4:450-455, 1993, the disclosure of which
is incorporated herein by reference in its entirety.
[0210] In some embodiments, the variants are created using
shuffling procedures wherein portions of a plurality of nucleic
acids which encode distinct polypeptides are fused together to
create chimeric nucleic acid sequences which encode chimeric
polypeptides as described in U.S. Pat. No.5,965,408, filed Jul. 9,
1996, entitled, "Method of DNA Reassembly by Interrupting
Synthesis", and U.S. Pat. No. 5,939,250, filed May 22, 1996,
entitled, "Production of Enzymes Having Desired Activities by
Mutagenesis", both of which are incorporated herein by
reference.
[0211] The variants of the polypeptides of Group B amino acid
sequences may be variants in which one or more of the amino acid
residues of the polypeptides of the Group B amino acid sequences
are substituted with a conserved or non-conserved amino acid
residue (preferably a conserved amino acid residue) and such
substituted amino acid residue may or may not be one encoded by the
genetic code.
[0212] Conservative substitutions are those that substitute a given
amino acid in a polypeptide by another amino acid of like
characteristics. Typically seen as conservative substitutions are
the following replacements: replacements of an aliphatic amino acid
such as Alanine, Valine, Leucine and Isoleucine with another
aliphatic amino acid; replacement of a Serine with a Threonine or
vice versa; replacement of an acidic residue such as Aspartic acid
and Glutamic acid with another acidic residue; replacement of a
residue bearing an amide group, such as Asparagine and Glutamine,
with another residue bearing an amide group; exchange of a basic
residue such as Lysine and Arginine with another basic residue; and
replacement of an aromatic residue such as Phenylalanine, Tyrosine
with another aromatic residue.
[0213] Other variants are those in which one or more of the amino
acid residues of the polypeptides of the Group B amino acid
sequences includes a substituent group.
[0214] Still other variants are those in which the polypeptide is
associated with another compound, such as a compound to increase
the half-life of the polypeptide (for example, polyethylene
glycol).
[0215] Additional variants are those in which additional amino
acids are fused to the polypeptide, such as a leader sequence, a
secretory sequence, a proprotein sequence or a sequence which
facilitates purification, enrichment, or stabilization of the
polypeptide.
[0216] In some embodiments, the fragments, derivatives and analogs
retain the same biological function or activity as the polypeptides
of Group B amino acid sequences, and sequences substantially
identical thereto. In other embodiments, the fragment, derivative,
or analog includes a proprotein, such that the fragment,
derivative, or analog can be activated by cleavage of the
proprotein portion to produce an active polypeptide.
[0217] Another aspect of the invention is polypeptides or fragments
thereof which have at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
more than about 95% homology to one of the polypeptides of Group B
amino acid sequences, and sequences substantially identical
thereto, or a fragment comprising at least 5, 10, 15, 20, 25, 30,
35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.
Homology may be determined using any of the programs described
above which aligns the polypeptides or fragments being compared and
determines the extent of amino acid identity or similarity between
them. It will be appreciated that amino acid "homology" includes
conservative amino acid substitutions such as those described
above.
[0218] The polypeptides or fragments having homology to one of the
polypeptides of Group B amino acid sequences, and sequences
substantially identical thereto, or a fragment comprising at least
about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids thereof may be obtained by isolating the
nucleic acids encoding them using the techniques described
above.
[0219] Alternatively, the homologous polypeptides or fragments may
be obtained through biochemical enrichment or purification
procedures. The sequence of potentially homologous polypeptides or
fragments may be determined by proteolytic digestion, gel
electrophoresis and/or microsequencing. The sequence of the
prospective homologous polypeptide or fragment can be compared to
one of the polypeptides of Group B amino acid sequences, and
sequences substantially identical thereto, or a fragment comprising
at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids thereof using any of the programs described
above.
[0220] Another aspect of the invention is an assay for identifying
fragments or variants of Group B amino acid sequences, and
sequences substantially identical thereto, which retain the
enzymatic function of the polypeptides of Group B amino acid
sequences, and sequences substantially identical thereto. For
example the fragments or variants of said polypeptides, may be used
to catalyze biochemical reactions, which indicate that the fragment
or variant retains the enzymatic activity of the polypeptides in
the Group B amino acid sequences.
[0221] The assay for determining if fragments of variants retain
the enzymatic activity of the polypeptides of Group B amino acid
sequences, and sequences substantially identical thereto includes
the steps of: contacting the polypeptide fragment or variant with a
substrate molecule under conditions which allow the polypeptide
fragment or variant to function, and detecting either a decrease in
the level of substrate or an increase in the level of the specific
reaction product of the reaction between the polypeptide and
substrate.
[0222] The polypeptides of Group B amino acid sequences, and
sequences substantially identical thereto or fragments comprising
at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids thereof may be used in a variety of
applications. For example, the polypeptides or fragments thereof
may be used to catalyze biochemical reactions. In accordance with
one aspect of the invention, there is provided a process for
utilizing the polypeptides of Group B amino acid sequences, and
sequences substantially identical thereto or polynucleotides
encoding such polypeptides for hydrolyzing glycosidic linkages. In
such procedures, a substance containing a glycosidic linkage (e.g.,
a starch) is contacted with one of the polypeptides of Group B
amino acid sequences, or sequences substantially identical thereto
under conditions which facilitate the hydrolysis of the glycosidic
linkage.
[0223] The polypeptides of Group B amino acid sequences, and
sequences substantially identical thereto or fragments comprising
at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids thereof, may also be used to generate
antibodies which bind specifically to the polypeptides or
fragments. The resulting antibodies may be used in immunoaffinity
chromatography procedures to isolate or purify the polypeptide or
to determine whether the polypeptide is present in a biological
sample. In such procedures, a protein preparation, such as an
extract, or a biological sample is contacted with an antibody
capable of specifically binding to one of the polypeptides of Group
B amino acid sequences, and sequences substantially identical
thereto, or fragments comprising at least 5, 10, 15, 20, 25, 30,
35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.
[0224] In immunoaffinity procedures, the antibody is attached to a
solid support, such as a bead or other column matrix. The protein
preparation is placed in contact with the antibody under conditions
in which the antibody specifically binds to one of the polypeptides
of Group B amino acid sequences, and sequences substantially
identical thereto, or fragment thereof. After a wash to remove
non-specifically bound proteins, the specifically bound
polypeptides are eluted.
[0225] The ability of proteins in a biological sample to bind to
the antibody may be determined using any of a variety of procedures
familiar to those skilled in the art. For example, binding may be
determined by labeling the antibody with a detectable label such as
a fluorescent agent, an enzymatic label, or a radioisotope.
Alternatively, binding of the antibody to the sample may be
detected using a secondary antibody having such a detectable label
thereon. Particular assays include ELISA assays, sandwich assays,
radioimmunoassays, and Western Blots.
[0226] Polyclonal antibodies generated against the polypeptides of
Group B amino acid sequences, and sequences substantially identical
thereto, or fragments comprising at least 5, 10, 15, 20, 25, 30,
35, 40, 50, 75, 100, or 150 consecutive amino acids thereof can be
obtained by direct injection of the polypeptides into an animal or
by administering the polypeptides to an animal, for example, a
nonhuman. The antibody so obtained will then bind the polypeptide
itself. In this manner, even a sequence encoding only a fragment of
the polypeptide can be used to generate antibodies which may bind
to the whole native polypeptide. Such antibodies can then be used
to isolate the polypeptide from cells expressing that
polypeptide.
[0227] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, Nature, 256:495-497, 1975, the disclosure of which is
incorporated herein by reference), the trioma technique, the human
B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72,
1983, the disclosure of which is incorporated herein by reference),
and the EBV-hybridoma technique (Cole, et al., 1985, in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, the
disclosure of which is incorporated herein by reference).
[0228] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778, the disclosure of which is
incorporated herein by reference) can be adapted to produce single
chain antibodies to the polypeptides of Group B amino acid
sequences, and sequences substantially identical thereto, or
fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50,
75, 100, or 150 consecutive amino acids thereof. Alternatively,
transgenic mice may be used to express humanized antibodies to
these polypeptides or fragments thereof.
[0229] Antibodies generated against the polypeptides of Group B
amino acid sequences, and sequences substantially identical
thereto, or fragments comprising at least 5, 10, 15, 20, 25, 30,
35, 40, 50, 75, 100, or 150 consecutive amino acids thereof may be
used in screening for similar polypeptides from other organisms and
samples. In such techniques, polypeptides from the organism are
contacted with the antibody and those polypeptides which
specifically bind the antibody are detected. Any of the procedures
described above may be used to detect antibody binding. One such
screening assay is described in "Methods for Measuring Cellulase
Activities", Methods in Enzymology, Vol 160, pp. 87-116, which is
hereby incorporated by reference in its entirety.
[0230] As used herein the term "nucleic acid sequence as set forth
in SEQ ID Nos.: 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40,
41, 43, 45, 47, 49, 51 and 53" encompasses the nucleotide sequences
of Group A nucleic acid sequences, and sequences substantially
identical thereto, as well as sequences homologous to Group A
nucleic acid sequences, and fragments thereof and sequences
complementary to all of the preceding sequences. The fragments
include portions of SEQ ID Nos.: 19, 20, 21, 22, 23, 24, 25, 26,
27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51 and 53 comprising at
least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400,
or 500 consecutive nucleotides of Group A nucleic acid sequences,
and sequences substantially identical thereto. Homologous sequences
and fragments of Group A nucleic acid sequences, and sequences
substantially identical thereto, refer to a sequence having at
least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,
55%, or 50% homology to these sequences. Homology may be determined
using any of the computer programs and parameters described herein,
including FASTA version 3.0t78 with the default parameters.
Homologous sequences also include RNA sequences in which uridines
replace the thymines in the nucleic acid sequences as set forth in
the Group A nucleic acid sequences. The homologous sequences may be
obtained using any of the procedures described herein or may result
from the correction of a sequencing error. It will be appreciated
that the nucleic acid sequences as set forth in Group A nucleic
acid sequences, and sequences substantially identical thereto, can
be represented in the traditional single character format (See the
inside back cover of Stryer, Lubert. Biochemistry, 3rd Ed., W. H
Freeman & Co., New York.) or in any other format which records
the identity of the nucleotides in a sequence.
[0231] As used herein the term "a polypeptide sequence as set forth
in SEQ ID Nos.:28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46,
47, 48, 50, 52, and 54" encompasses the polypeptide sequence of
Group B amino acid sequences, and sequences substantially identical
thereto, which are encoded by a sequence as set forth in SEQ ID
Nos.: 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43,
45, 47, 49, 51, 53, polypeptide sequences homologous to the
polypeptides of Group B amino acid sequences, and sequences
substantially identical thereto, or fragments of any of the
preceding sequences. Homologous polypeptide sequences refer to a
polypeptide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,
85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% homology to one of the
polypeptide sequences of the Group B amino acid sequences. Homology
may be determined using any of the computer programs and parameters
described herein, including FASTA version 3.0t78 with the default
parameters or with any modified parameters. The homologous
sequences may be obtained using any of the procedures described
herein or may result from the correction of a sequencing error. The
polypeptide fragments comprise at least 5, 10, 15, 20, 25, 30, 35,
40, 50, 75, 100, or 150 consecutive amino acids of the polypeptides
of Group B amino acid sequences, and sequences substantially
identical thereto. It will be appreciated that the polypeptide
codes as set forth in Group B amino acid sequences, and sequences
substantially identical thereto, can be represented in the
traditional single character format or three letter format (See the
inside back cover of Stryer, Lubert. Biochemistry, 3rd Ed., W. H
Freeman & Co., New York.) or in any other format which relates
the identity of the polypeptides in a sequence.
[0232] It will be appreciated by those skilled in the art that a
nucleic acid sequence as set forth in SEQ ID Nos.: 19, 20, 21, 22,
23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, and 53
and a polypeptide sequence as set forth in SEQ ID Nos.:28, 29, 30,
31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, and 54 can
be stored, recorded, and manipulated on any medium which can be
read and accessed by a computer. As used herein, the words
"recorded" and "stored" refer to a process for storing information
on a computer medium. A skilled artisan can readily adopt any of
the presently known methods for recording information on a computer
readable medium to generate manufactures comprising one or more of
the nucleic acid sequences as set forth in Group A nucleic acid
sequences, and sequences substantially identical thereto, one or
more of the polypeptide sequences as set forth in Group B amino
acid sequences, and sequences substantially identical thereto.
Another aspect of the invention is a computer readable medium
having recorded thereon at least 2, 5, 10, 15, or 20 nucleic acid
sequences as set forth in Group A nucleic acid sequences, and
sequences substantially identical thereto.
[0233] Another aspect of the invention is a computer readable
medium having recorded thereon one or more of the nucleic acid
sequences as set forth in Group A nucleic acid sequences, and
sequences substantially identical thereto. Another aspect of the
invention is a computer readable medium having recorded thereon one
or more of the polypeptide sequences as set forth in Group B amino
acid sequences, and sequences substantially identical thereto.
Another aspect of the invention is a computer readable medium
having recorded thereon at least 2, 5, 10, 15, or 20 of the
sequences as set forth above.
[0234] Computer readable media include magnetically readable media,
optically readable media, electronically readable media and
magnetic/optical media. For example, the computer readable media
may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital
Versatile Disk (DVD), Random Access Memory (RAM), or Read Only
Memory (ROM) as well as other types of other media known to those
skilled in the art.
[0235] Embodiments of the invention include systems (e.g., internet
based systems), particularly computer systems which store and
manipulate the sequence information described herein. One example
of a computer system 100 is illustrated in block diagram form in
FIG. 1. As used herein, "a computer system" refers to the hardware
components, software components, and data storage components used
to analyze a nucleotide sequence of a nucleic acid sequence as set
forth in Group A nucleic acid sequences, and sequences
substantially identical thereto, or a polypeptide sequence as set
forth in the Group B amino acid sequences. The computer system 100
typically includes a processor for processing, accessing and
manipulating the sequence data. The processor 105 can be any
well-known type of central processing unit, such as, for example,
the Pentium III from Intel Corporation, or similar processor from
Sun, Motorola, Compaq, AMD or International Business Machines.
[0236] Typically the computer system 100 is a general purpose
system that comprises the processor 105 and one or more internal
data storage components 110 for storing data, and one or more data
retrieving devices for retrieving the data stored on the data
storage components. A skilled artisan can readily appreciate that
any one of the currently available computer systems are
suitable.
[0237] In one particular embodiment, the computer system 100
includes a processor 105 connected to a bus which is connected to a
main memory 115 (preferably implemented as RAM) and one or more
internal data storage devices 110, such as a hard drive and/or
other computer readable media having data recorded thereon. In some
embodiments, the computer system 100 further includes one or more
data retrieving device 118 for reading the data stored on the
internal data storage devices 110.
[0238] The data retrieving device 118 may represent, for example, a
floppy disk drive, a compact disk drive, a magnetic tape drive, or
a modem capable of connection to a remote data storage system
(e.g., via the internet) etc. In some embodiments, the internal
data storage device 110 is a removable computer readable medium
such as a floppy disk, a compact disk, a magnetic tape, etc.
containing control logic and/or data recorded thereon. The computer
system 100 may advantageously include or be programmed by
appropriate software for reading the control logic and/or the data
from the data storage component once inserted in the data
retrieving device.
[0239] The computer system 100 includes a display 120 which is used
to display output to a computer user. It should also be noted that
the computer system 100 can be linked to other computer systems
125a-c in a network or wide area network to provide centralized
access to the computer system 100.
[0240] Software for accessing and processing the nucleotide
sequences of a nucleic acid sequence as set forth in Group A
nucleic acid sequences, and sequences substantially identical
thereto, or a polypeptide sequence as set forth in Group B amino
acid sequences, and sequences substantially identical thereto,
(such as search tools, compare tools, and modeling tools etc.) may
reside in main memory 115 during execution.
[0241] In some embodiments, the computer system 100 may further
comprise a sequence comparison algorithm for comparing a nucleic
acid sequence as set forth in Group A nucleic acid sequences, and
sequences substantially identical thereto, or a polypeptide
sequence as set forth in Group B amino acid sequences, and
sequences substantially identical thereto, stored on a computer
readable medium to a reference nucleotide or polypeptide
sequence(s) stored on a computer readable medium. A "sequence
comparison algorithm" refers to one or more programs which are
implemented (locally or remotely) on the computer system 100 to
compare a nucleotide sequence with other nucleotide sequences
and/or compounds stored within a data storage means. For example,
the sequence comparison algorithm may compare the nucleotide
sequences of a nucleic acid sequence as set forth in Group A
nucleic acid sequences, and sequences substantially identical
thereto, or a polypeptide sequence as set forth in Group B amino
acid sequences, and sequences substantially identical thereto,
stored on a computer readable medium to reference sequences stored
on a computer readable medium to identify homologies or structural
motifs. Various sequence comparison programs identified elsewhere
in this patent specification are particularly contemplated for use
in this aspect of the invention. Protein and/or nucleic acid
sequence homologies may be evaluated using any of the variety of
sequence comparison algorithms and programs known in the art. Such
algorithms and programs include, but are by no means limited to,
TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman,
Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al.,
J. Mol. Biol. 215(3):403-410, 1990; Thompson et al., Nucleic Acids
Res. 22(2):4673-4680, 1994; Higgins et al., Methods Enzymol.
266:383-402, 1996; Altschul et al., J. Mol. Biol. 215(3):403-410,
1990; Altschul et al., Nature Genetics 3:266-272, 1993).
[0242] Homology or identity is often measured using sequence
analysis software (e.g., Sequence Analysis Software Package of the
Genetics Computer Group, University of Wisconsin Biotechnology
Center, 1710 University Avenue, Madison, Wis. 53705). Such software
matches similar sequences by assigning degrees of homology to
various deletions, substitutions and other modifications. The terms
"homology" and "identity" in the context of two or more nucleic
acids or polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues or nucleotides that are the same when compared
and aligned for maximum correspondence over a comparison window or
designated region as measured using any number of sequence
comparison algorithms or by manual alignment and visual
inspection.
[0243] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0244] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequence for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol 48:443, 1970, by
the search for similarity method of person & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection. Other algorithms for determining
homology or identity include, for example, in addition to a BLAST
program (Basic Local Alignment Search Tool at the National Center
for Biological Information), ALIGN, AMAS (Analysis of Multiply
Aligned Sequences), AMPS (Protein Multiple Sequence Alignment),
ASSET (Aligned Segment Statistical Evaluation Tool), BANDS,
BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node),
BLIMPS (BLocks IMProved Searcher), FASTA, Intervals & Points,
BMB, CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS,
Smith-Waterman algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced
Nucleotide Alignment Tool), Framealign, Framesearch, DYNAMIC,
FILTER, FSAP (Fristensky Sequence Analysis Package), GAP (Global
Alignment Program), GENAL, GIBBS, GenQuest, ISSC (Sensitive
Sequence Comparison), LALIGN (Local Sequence Alignment), LCP (Local
Content Program), MACAW (Multiple Alignment Construction &
Analysis Workbench), MAP (Multiple Alignment Program), MBLKP,
MBLKN, PIMA (Pattern-Induced Multi-sequence Alignment), SAGA
(Sequence Alignment by Genetic Algorithm) and WHAT-IF. Such
alignment programs can also be used to screen genome databases to
identify polynucleotide sequences having substantially identical
sequences. A number of genome databases are available, for example,
a substantial portion of the human genome is available as part of
the Human Genome Sequencing Project (J. Roach,
http://weber.u.Washingt- on.edu/.about.roach/human_genome_progress
2.html) (Gibbs, 1995). At least twenty-one other genomes have
already been sequenced, including, for example, M. genitalium
(Fraser et al., 1995), M. jannaschii (Bult et al., 1996), H.
influenzae (Fleischmann et al., 1995), E. coli (Blattner et al.,
1997), and yeast (S. cerevisiae) (Mewes et al., 1997), and D.
melanogaster (Adams et al., 2000). Significant progress has also
been made in sequencing the genomes of model organism, such as
mouse, C. elegans, and Arabadopsis sp. Several databases containing
genomic information annotated with some functional information are
maintained by different organization, and are accessible via the
internet, for example, http://wwwtigr.org/tdb;
http://www.genetics.wisc.edu; http://genome-www.stanford.edu/-ball;
http://hiv-web.lanl.gov; http://www.ncbi.nlm.nih.gov;
http://www.ebi.ac.uk; http://Pasteur.fr/other/biology; and
http://www.genome.wi.mit.edu.
[0245] One example of a useful algorithm is BLAST and BLAST 2.0
algorithms, which are described in Altschul et al., Nuc. Acids Res.
25:3389-3402, 1977, and Altschul et al., J. Mol. Biol. 215:403-410,
1990, respectively. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match
or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to
as the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0). For
amino acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectations (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915, 1989) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0246] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Natl. Acad. Sci. USA 90:5873, 1993). One measure of
similarity provided by BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a references sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.2, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0247] In one embodiment, protein and nucleic acid sequence
homologies are evaluated using the Basic Local Alignment Search
Tool ("BLAST") In particular, five specific BLAST programs are used
to perform the following task:
[0248] (1) BLASTP and BLAST3 compare an amino acid query sequence
against a protein sequence database;
[0249] (2) BLASTN compares a nucleotide query sequence against a
nucleotide sequence database;
[0250] (3) BLASTX compares the six-frame conceptual translation
products of a query nucleotide sequence (both strands) against a
protein sequence database;
[0251] (4) TBLASTN compares a query protein sequence against a
nucleotide sequence database translated in all six reading frames
(both strands); and
[0252] (5) TBLASTX compares the six-frame translations of a
nucleotide query sequence against the six-frame translations of a
nucleotide sequence database.
[0253] The BLAST programs identify homologous sequences by
identifying similar segments, which are referred to herein as
"high-scoring segment pairs," between a query amino or nucleic acid
sequence and a test sequence which is preferably obtained from a
protein or nucleic acid sequence database. High-scoring segment
pairs are preferably identified (i.e., aligned) by means of a
scoring matrix, many of which are known in the art. Preferably, the
scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science
256:1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61,
1993). Less preferably, the PAM or PAM250 matrices may also be used
(see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for
Detecting Distance Relationships: Atlas of protein Sequence and
Structure, Washington: National Biomedical Research Foundation).
BLAST programs are accessible through the U.S. National Library of
Medicine, e.g., at www.ncbi.nlm.nih.gov.
[0254] The parameters used with the above algorithms may be adapted
depending on the sequence length and degree of homology studied. In
some embodiments, the parameters may be the default parameters used
by the algorithms in the absence of instructions from the user.
[0255] FIG. 2 is a flow diagram illustrating one embodiment of a
process 200 for comparing a new nucleotide or protein sequence with
a database of sequences in order to determine the homology levels
between the new sequence and the sequences in the database. The
database of sequences can be a private database stored within the
computer system 100, or a public database such as GENBANK that is
available through the Internet.
[0256] The process 200 begins at a start state 201 and then moves
to a state 202 wherein the new sequence to be compared is stored to
a memory in a computer system 100. As discussed above, the memory
could be any type of memory, including RAM or an internal storage
device.
[0257] The process 200 then moves to a state 204 wherein a database
of sequences is opened for analysis and comparison. The process 200
then moves to a state 206 wherein the first sequence stored in the
database is read into a memory on the computer. A comparison is
then performed at a state 210 to determine if the first sequence is
the same as the second sequence. It is important to note that this
step is not limited to performing an exact comparison between the
new sequence and the first sequence in the database. Well-known
methods are known to those of skill in the art for comparing two
nucleotide or protein sequences, even if they are not identical.
For example, gaps can be introduced into one sequence in order to
raise the homology level between the two tested sequences. The
parameters that control whether gaps or other features are
introduced into a sequence during comparison are normally entered
by the user of the computer system.
[0258] Once a comparison of the two sequences has been performed at
the state 210, a determination is made at a decision state 210
whether the two sequences are the same. Of course, the term "same"
is not limited to sequences that are absolutely identical.
Sequences that are within the homology parameters entered by the
user will be marked as "same" in the process 200.
[0259] If a determination is made that the two sequences are the
same, the process 200 moves to a state 214 wherein the name of the
sequence from the database is displayed to the user. This state
notifies the user that the sequence with the displayed name
fulfills the homology constraints that were entered. Once the name
of the stored sequence is displayed to the user, the process 200
moves to a decision state 218 wherein a determination is made
whether more sequences exist in the database. If no more sequences
exist in the database, then the process 200 terminates at an end
state 220. However, if more sequences do exist in the database,
then the process 200 moves to a state 224 wherein a pointer is
moved to the next sequence in the database so that it can be
compared to the new sequence. In this manner, the new sequence is
aligned and compared with every sequence in the database.
[0260] It should be noted that if a determination had been made at
the decision state 212 that the sequences were not homologous, then
the process 200 would move immediately to the decision state 218 in
order to determine if any other sequences were available in the
database for comparison.
[0261] Accordingly, one aspect of the invention is a computer
system comprising a processor, a data storage device having stored
thereon a nucleic acid sequence as set forth in Group A nucleic
acid sequences, and sequences substantially identical thereto, or a
polypeptide sequence as set forth in Group B amino acid sequences,
and sequences substantially identical thereto, a data storage
device having retrievably stored thereon reference nucleotide
sequences or polypeptide sequences to be compared to a nucleic acid
sequence as set forth in Group A nucleic acid sequences, and
sequences substantially identical thereto, or a polypeptide
sequence as set forth in Group B amino acid sequences, and
sequences substantially identical thereto, and a sequence comparer
for conducting the comparison. The sequence comparer may indicate a
homology level between the sequences compared or identify
structural motifs in the above described nucleic acid code of Group
A nucleic acid sequences, and sequences substantially identical
thereto, or a polypeptide sequence as set forth in Group B amino
acid sequences, and sequences substantially identical thereto, or
it may identify structural motifs in sequences which are compared
to these nucleic acid codes and polypeptide codes. In some
embodiments, the data storage device may have stored thereon the
sequences of at least 2, 5, 10, 15, 20, 25, 30 or 40 or more of the
nucleic acid sequences as set forth in Group A nucleic acid
sequences, and sequences substantially identical thereto, or the
polypeptide sequences as set forth in Group B amino acid sequences,
and sequences substantially identical thereto.
[0262] Another aspect of the invention is a method for determining
the level of homology between a nucleic acid sequence as set forth
in Group A nucleic acid sequences, and sequences substantially
identical thereto, or a polypeptide sequence as set forth in Group
B amino acid sequences, and sequences substantially identical
thereto, and a reference nucleotide sequence. The method including
reading the nucleic acid code or the polypeptide code and the
reference nucleotide or polypeptide sequence through the use of a
computer program which determines homology levels and determining
homology between the nucleic acid code or polypeptide code and the
reference nucleotide or polypeptide sequence with the computer
program. The computer program may be any of a number of computer
programs for determining homology levels, including those
specifically enumerated herein, (e.g., BLAST2N with the default
parameters or with any modified parameters). The method may be
implemented using the computer systems described above. The method
may also be performed by reading at least 2, 5, 10, 15, 20, 25, 30
or 40 or more of the above described nucleic acid sequences as set
forth in the Group A nucleic acid sequences, or the polypeptide
sequences as set forth in the Group B amino acid sequences through
use of the computer program and determining homology between the
nucleic acid codes or polypeptide codes and reference nucleotide
sequences or polypeptide sequences.
[0263] FIG. 3 is a flow diagram illustrating one embodiment of a
process 250 in a computer for determining whether two sequences are
homologous. The process 250 begins at a start state 252 and then
moves to a state 254 wherein a first sequence to be compared is
stored to a memory. The second sequence to be compared is then
stored to a memory at a state 256. The process 250 then moves to a
state 260 wherein the first character in the first sequence is read
and then to a state 262 wherein the first character of the second
sequence is read. It should be understood that if the sequence is a
nucleotide sequence, then the character would normally be either A,
T, C, G or U. If the sequence is a protein sequence, then it is
preferably in the single letter amino acid code so that the first
and sequence sequences can be easily compared.
[0264] A determination is then made at a decision state 264 whether
the two characters are the same. If they are the same, then the
process 250 moves to a state 268 wherein the next characters in the
first and second sequences are read. A determination is then made
whether the next characters are the same. If they are, then the
process 250 continues this loop until two characters are not the
same. If a determination is made that the next two characters are
not the same, the process 250 moves to a decision state 274 to
determine whether there are any more characters either sequence to
read.
[0265] If there are not any more characters to read, then the
process 250 moves to a state 276 wherein the level of homology
between the first and second sequences is displayed to the user.
The level of homology is determined by calculating the proportion
of characters between the sequences that were the same out of the
total number of sequences in the first sequence. Thus, if every
character in a first 100 nucleotide sequence aligned with a every
character in a second sequence, the homology level would be
100%.
[0266] Alternatively, the computer program may be a computer
program which compares the nucleotide sequences of a nucleic acid
sequence as set forth in the invention, to one or more reference
nucleotide sequences in order to determine whether the nucleic acid
code of Group A nucleic acid sequences, and sequences substantially
identical thereto, differs from a reference nucleic acid sequence
at one or more positions. Optionally such a program records the
length and identity of inserted, deleted or substituted nucleotides
with respect to the sequence of either the reference polynucleotide
or a nucleic acid sequence as set forth in Group A nucleic acid
sequences, and sequences substantially identical thereto. In one
embodiment, the computer program may be a program which determines
whether a nucleic acid sequence as set forth in Group A nucleic
acid sequences, and sequences substantially identical thereto,
contains a single nucleotide polymorphism (SNP) with respect to a
reference nucleotide sequence.
[0267] Accordingly, another aspect of the invention is a method for
determining whether a nucleic acid sequence as set forth in Group A
nucleic acid sequences, and sequences substantially identical
thereto, differs at one or more nucleotides from a reference
nucleotide sequence comprising the steps of reading the nucleic
acid code and the reference nucleotide sequence through use of a
computer program which identifies differences between nucleic acid
sequences and identifying differences between the nucleic acid code
and the reference nucleotide sequence with the computer program. In
some embodiments, the computer program is a program which
identifies single nucleotide polymorphisms. The method may be
implemented by the computer systems described above and the method
illustrated in FIG. 3. The method may also be performed by reading
at least 2, 5, 10, 15, 20, 25, 30, or 40 or more of the nucleic
acid sequences as set forth in Group A nucleic acid sequences, and
sequences substantially identical thereto, and the reference
nucleotide sequences through the use of the computer program and
identifying differences between the nucleic acid codes and the
reference nucleotide sequences with the computer program.
[0268] In other embodiments the computer based system may further
comprise an identifier for identifying features within a nucleic
acid sequence as set forth in the Group A nucleic acid sequences or
a polypeptide sequence as set forth in Group B amino acid
sequences, and sequences substantially identical thereto.
[0269] An "identifier" refers to one or more programs which
identifies certain features within a nucleic acid sequence as set
forth in Group A nucleic acid sequences, and sequences
substantially identical thereto, or a polypeptide sequence as set
forth in Group B amino acid sequences, and sequences substantially
identical thereto. In one embodiment, the identifier may comprise a
program which identifies an open reading frame in a nucleic acid
sequence as set forth in Group A nucleic acid sequences, and
sequences substantially identical thereto.
[0270] FIG. 5 is a flow diagram illustrating one embodiment of an
identifier process 300 for detecting the presence of a feature in a
sequence. The process 300 begins at a start state 302 and then
moves to a state 304 wherein a first sequence that is to be checked
for features is stored to a memory 115 in the computer system 100.
The process 300 then moves to a state 306 wherein a database of
sequence features is opened. Such a database would include a list
of each feature's attributes along with the name of the feature.
For example, a feature name could be "Initiation Codon" and the
attribute would be "ATG". Another example would be the feature name
"TAATAA Box" and the feature attribute would be "TAATAA". An
example of such a database is produced by the University of
Wisconsin Genetics Computer Group (www.gcg.com). Alternatively, the
features may be structural polypeptide motifs such as alpha
helices, beta sheets, or functional polypeptide motifs such as
enzymatic active sites, helix-turn-helix motifs or other motifs
known to those skilled in the art.
[0271] Once the database of features is opened at the state 306,
the process 300 moves to a state 308 wherein the first feature is
read from the database. A comparison of the attribute of the first
feature with the first sequence is then made at a state 310. A
determination is then made at a decision state 316 whether the
attribute of the feature was found in the first sequence. If the
attribute was found, then the process 300 moves to a state 318
wherein the name of the found feature is displayed to the user.
[0272] The process 300 then moves to a decision state 320 wherein a
determination is made whether move features exist in the database.
If no more features do exist, then the process 300 terminates at an
end state 324. However, if more features do exist in the database,
then the process 300 reads the next sequence feature at a state 326
and loops back to the state 310 wherein the attribute of the next
feature is compared against the first sequence.
[0273] It should be noted, that if the feature attribute is not
found in the first sequence at the decision state 316, the process
300 moves directly to the decision state 320 in order to determine
if any more features exist in the database.
[0274] Accordingly, another aspect of the invention is a method of
identifying a feature within a nucleic acid sequence as set forth
in Group A nucleic acid sequences, and sequences substantially
identical thereto, or a polypeptide sequence as set forth in Group
B amino acid sequences, and sequences substantially identical
thereto, comprising reading the nucleic acid code(s) or polypeptide
code(s) through the use of a computer program which identifies
features therein and identifying features within the nucleic acid
code(s) with the computer program. In one embodiment, computer
program comprises a computer program which identifies open reading
frames. The method may be performed by reading a single sequence or
at least 2, 5, 10, 15, 20, 25, 30, or 40 of the nucleic acid
sequences as set forth in Group A nucleic acid sequences, and
sequences substantially identical thereto, or the polypeptide
sequences as set forth in Group B amino acid sequences, and
sequences substantially identical thereto, through the use of the
computer program and identifying features within the nucleic acid
codes or polypeptide codes with the computer program.
[0275] A nucleic acid sequence as set forth in Group A nucleic acid
sequences, and sequences substantially identical thereto, or a
polypeptide sequence as set forth in Group B amino acid sequences,
and sequences substantially identical thereto, may be stored and
manipulated in a variety of data processor programs in a variety of
formats. For example, a nucleic acid sequence as set forth in Group
A nucleic acid sequences, and sequences substantially identical
thereto, or a polypeptide sequence as set forth in Group B amino
acid sequences, and sequences substantially identical thereto, may
be stored as text in a word processing file, such as MicrosoftWORD
or WORDPERFECT or as an ASCII file in a variety of database
programs familiar to those of skill in the art, such as DB2,
SYBASE, or ORACLE. In addition, many computer programs and
databases may be used as sequence comparison algorithms,
identifiers, or sources of reference nucleotide sequences or
polypeptide sequences to be compared to a nucleic acid sequence as
set forth in Group A nucleic acid sequences, and sequences
substantially identical thereto, or a polypeptide sequence as set
forth in Group B amino acid sequences, and sequences substantially
identical thereto. The following list is intended not to limit the
invention but to provide guidance to programs and databases which
are useful with the nucleic acid sequences as set forth in Group A
nucleic acid sequences, and sequences substantially identical
thereto, or the polypeptide sequences as set forth in Group B amino
acid sequences, and sequences substantially identical thereto.
[0276] The programs and databases which may be used include, but
are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular
Applications Group), GeneMine (Molecular Applications Group), Look
(Molecular Applications Group), MacLook (Molecular Applications
Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al,
J. Mol. Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc.
Natl. Acad. Sci. USA, 85: 2444, 1988), FASTDB (Brutlag et al. Comp.
App. Biosci. 6:237-245, 1990), Catalyst (Molecular Simulations
Inc.), Catalyst/SHAPE (Molecular Simulations Inc.),
Cerius.sup.2.DBAccess (Molecular Simulations Inc.), HypoGen
(Molecular Simulations Inc.), Insight II, (Molecular Simulations
Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular
Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi,
(Molecular Simulations Inc.), QuanteMM, (Molecular Simulations
Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular
Simulations Inc.), ISIS (Molecular Simulations Inc.),
Quanta/Protein Design (Molecular Simulations Inc.), WebLab
(Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular
Simulations Inc.), Gene Explorer (Molecular Simulations Inc.),
SeqFold (Molecular Simulations Inc.), the MDL Available Chemicals
Directory database, the MDL Drug Data Report data base, the
Comprehensive Medicinal Chemistry database, Derwent's World Drug
Index database, the BioByteMasterFile database, the Genbank
database, and the Genseqn database. Many other programs and data
bases would be apparent to one of skill in the art given the
present disclosure.
[0277] Motifs which may be detected using the above programs
include sequences encoding leucine zippers, helix-turn-helix
motifs, glycosylation sites, ubiquitination sites, alpha helices,
and beta sheets, signal sequences encoding signal peptides which
direct the secretion of the encoded proteins, sequences implicated
in transcription regulation such as homeoboxes, acidic stretches,
enzymatic active sites, substrate binding sites, and enzymatic
cleavage sites.
[0278] The present invention exploits the unique catalytic
properties of enzymes. Whereas the use of biocatalysts (i.e.,
purified or crude enzymes, non-living or living cells) in chemical
transformations normally requires the identification of a
particular biocatalyst that reacts with a specific starting
compound, the present invention uses selected biocatalysts and
reaction conditions that are specific for functional groups that
are present in many starting compounds, such as small molecules.
Each biocatalyst is specific for one functional group, or several
related functional groups, and can react with many starting
compounds containing this functional group.
[0279] The biocatalytic reactions produce a population of
derivatives from a single starting compound. These derivatives can
be subjected to another round of biocatalytic reactions to produce
a second population of derivative compounds. Thousands of
variations of the original small molecule or compound can be
produced with each iteration of biocatalytic derivatization.
[0280] Enzymes react at specific sites of a starting compound
without affecting the rest of the molecule, a process which is very
difficult to achieve using traditional chemical methods. This high
degree of biocatalytic specificity provides the means to identify a
single active compound within the library. The library is
characterized by the series of biocatalytic reactions used to
produce it, a so called "biosynthetic history". Screening the
library for biological activities and tracing the biosynthetic
history identifies the specific reaction sequence producing the
active compound. The reaction sequence is repeated and the
structure of the synthesized compound determined. This mode of
identification, unlike other synthesis and screening approaches,
does not require immobilization technologies, and compounds can be
synthesized and tested free in solution using virtually any type of
screening assay. It is important to note, that the high degree of
specificity of enzyme reactions on functional groups allows for the
"tracking" of specific enzymatic reactions that make up the
biocatalytically produced library.
[0281] Many of the procedural steps are performed using robotic
automation enabling the execution of many thousands of biocatalytic
reactions and screening assays per day as well as ensuring a high
level of accuracy and reproducibility. As a result, a library of
derivative compounds can be produced in a matter of weeks which
would take years to produce using current chemical methods.
[0282] In a particular embodiment, the invention provides a method
for modifying small molecules, comprising contacting a polypeptide
encoded by a polynucleotide described herein or enzymatically
active fragments thereof with a small molecule to produce a
modified small molecule. A library of modified small molecules is
tested to determine if a modified small molecule is present within
the library which exhibits a desired activity. A specific
biocatalytic reaction which produces the modified small molecule of
desired activity is identified by systematically eliminating each
of the biocatalytic reactions used to produce a portion of the
library, and then testing the small molecules produced in the
portion of the library for the presence or absence of the modified
small molecule with the desired activity. The specific biocatalytic
reactions which produce the modified small molecule of desired
activity is optionally repeated. The biocatalytic reactions are
conducted with a group of biocatalysts that react with distinct
structural moieties found within the structure of a small molecule,
each biocatalyst is specific for one structural moiety or a group
of related structural moieties; and each biocatalyst reacts with
many different small molecules which contain the distinct
structural moiety.
[0283] The invention will be further described with reference to
the following examples; however, it is to be understood that the
invention is not limited to such examples.
EXAMPLES
Example 1
Bacterial Expression and-Purification of Alkaline Phosphatase
Enzymes
[0284] DNA encoding the enzymes of the present invention, SEQ ID
NOS:1 through 16, were initially amplified from a pBluescript
vector containing the DNA by the PCR technique using the primers
noted herein. The amplified sequences were then inserted into the
respective pQE vector listed beneath the primer sequences, and the
enzyme was expressed according to the protocols set forth herein.
The 5' and 3' oligonucleotide primer sequences used for subcloning
and vectors for the respective genes are as follows:
[0285] Ammonifex degensii KC4-3A1A
1 5' CCGA GAA TTC ATT AAA GAG GAG AAA TTA ACT ATG GGG GCA GGT CCG
AAA AGG 3' 5' CGGA GGA TCC CTA CAG TTC TAA AAG TCT TTT A 3' Vector:
pQET3
[0286] Methanococcus igneus Ko15-9A1A
2 5' CCGA GAA TTC ATT AAA GAG GAG AAA TTA ACT ATG TTG GAT ATA CTG
GIT 3' 5' CCGA CGA TCC TTA TTT TTT AAC CAA ATGT TCC 3' Vector:
pQET3
[0287] Thermococcus Alcaliphilus AEDII12RA-18A
3 5' CCGA CAA TTG ATT AAA GAG GAG AAA TTA ACT ATG ATG ATG GAA TTC
ACT CGC 3' 5' CGGA GGA TCC CTA CAG TTC TAA AAG TCT TIT A 3' Vector:
pQET3
[0288] Thermococcus Celer 25A1A (incorporating Mfe1 restriction
site)
4 5' CCGA CAA TTG ATT AAA GAG GAG AAA TTA ACT ATG AGA ACC CTG ACA
ATA AAC 3' 5' CCGA GGA TCC TTA CAC CCA CAG AAC CCT TAC 3' Vector
pQET3
[0289] Thermococcus GU5L5-26A1A
5 5' CCGA GAA TTC ATT AAA GAG GAG AAA TTA ACT ATG AAA GGA AAG TCT
CIT GIT 3' 5' CCGA GGA TCC TCA AGC TTC CTG GAG AAT CAA 3' Vector
pQET3
[0290] OC9a-27A3A
6 5' CCGA GAA TTC ATT AAA GAG GAG AAA TTA ACT ATG CCA AGA AAT ATC
GCC GCT 3' 5' CCGA GGA TCC TTA AGG CIT CTC GAG GTG GGG GIT 3'
Vector pQET3
[0291] M11 TL-29A1A (incorporating Mfel restriction site)
7 5' CCGA CAA TTG ATT AAA GAG GAG AAA TTA ACT ATG TAT AAA TGG ATT
ATT GAG GG 3' 5' CCGA GGA CTA AAC ATA GTC TAA, GTA ATT AGC 3'
Vector pQET3
[0292] Thermococcus CL-2 30A1A
8 5' CCGA GAA TTC ATT AAA GAG GAG AAA TTA ACT ATG AGA ATC CTC CTC
ACC AAC 3' 5' CCGA GGA TCC TCA CAG GCT CAG AAG CCT TTG 3' Vector
pQET3
[0293] Aquifex VF-5-34A1A
9 5' CCGA GAA TTC ATT AAA GAG GAG AAA TTA ACT ATG GAA AAC TTA AAA
AAG TAC CT 3' 5' CCGA GGA TCC TCA CCG CCC CCT GCG GGT GCG 3' Vector
pQET3
[0294] The restriction enzyme sites indicated correspond to the
restriction enzyme sites on the bacterial expression vector
indicated for the respective gene (Qiagen, Inc. Chatsworth,
Calif.). The pQE vector encodes antibiotic resistance (Amp.sup.r),
a bacterial origin of replication (ori), an IPTG-regulatable
promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag
and restriction enzyme sites.
[0295] The pQE vector was digested with the restriction enzymes
indicated. The amplified sequences were ligated into the respective
pQE vector and inserted in frame with the sequence encoding for the
RBS. The native stop codon was incorporated so the genes were not
fused to the His tag of the vector. The ligation mixture was then
used to transform the E. coli strain M15/pREP4 (Qiagen, Inc.) by
electroporation. M15/pREP4 contains multiple copies of the plasmid
pREP4, which expresses the lacI repressor and also confers
kanamycin resistance (Kan.sup.r). Transformants were identified by
their ability to grow on LB plates and ampicillin/kanamycin
resistant colonies were selected. Plasmid DNA was isolated and
confirmed by restriction analysis. Clones containing the desired
constructs were grown overnight (O/N) in liquid culture in LB media
supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N
culture was used to inoculate a large culture at a ratio of 1:100
to 1:250. The cells were grown to an optical density 600
(O.D..sup.600) of between 0.4 and 0.6. IPTG
("Isopropyl-B-D-thiogalacto pyranoside") was then added to a final
concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/O leading to increased gene expression.
Cells were grown an extra 3 to 4 hours. Cells were then harvested
by centrifugation.
[0296] The primer sequences set out above may also be employed to
isolate the target gene from the deposited material by
hybridization techniques described above.
Example 2
Isolation of A Selected-Clone From the Deposited Genomic Clones
[0297] A clone is isolated directly by screening the deposited
material using the oligonucleotide primers set forth in Example 1
for the particular gene desired to be isolated. The specific
oligonucleotides are synthesized using an Applied Biosystems DNA
synthesizer.
[0298] The two oligonucleotide primers corresponding to the gene of
interest are used to amplify the gene from the deposited material.
A polymerase chain reaction is carried out in 25 .mu.l of reaction
mixture with 0.1 ug of the DNA of the gene of interest. The
reaction mixture is 1.5-5 mM MgCl.sub.2, 0.01% (w/v) gelatin, 20
.mu.M each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and
1.25 Unit of Taq polymerase. Thirty cycles of PCR (denaturation at
94.degree. C. for 1 min; annealing at 55.degree. C. for 1 min;
elongation at 72.degree. C. for 1 min) are performed with the
Perkin-Elmer Cetus 9600 thermal cycler. The amplified product is
analyzed by agarose gel electrophoresis and the DNA band with
expected molecular weight is excised and purified. The PCR product
is verified to be the gene of interest by subcloning and sequencing
the DNA product. The ends of the newly purified genes are
nucleotide sequenced to identify full length sequences. Complete
sequencing of full length genes is then performed by Exonuclease
III digestion or primer walking.
[0299] While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
Sequence CWU 1
1
* * * * *
References